In situ combinatorial labeling of cellular molecules

ABSTRACT

Methods of uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue are provided. Kits for uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue are also provided. The molecules to be labeled may include, but are not limited to, RNAs and/or cDNAs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage entry of International PatentApplication no. PCT/US2018/052283, filed Sep. 21, 2018, which claims thebenefit of U.S. Provisional Application No. 62/561,806, filed Sep. 22,2017, which are both hereby incorporated by reference in their entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. R01CA207029, awarded by the National Institutes of Health. The governmenthas certain ridhts in the invention.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing is3915_P1047USPNPUW_Seq_List_FINAL_20220610_ST25.txt. The text file is12.0 KB; was created on Jun. 10, 2022, 2022; and is being submitted viaEFSWeb with the filing of the specification.

TECHNICAL FIELD

The present disclosure relates generally to methods of uniquely labelingor barcoding molecules within a nucleus, a plurality of nuclei, a cell,a plurality of cells, and/or a tissue. The present disclosure alsorelates to kits for uniquely labeling molecules within a nucleus, aplurality of nuclei, a cell, a plurality of cells, and/or a tissue. Inparticular, the methods and kits may relate to the labeling of RNAsand/or cDNAs.

BACKGROUND

Next Generation Sequencing (NGS) can be used to identify and/or quantifyindividual transcripts from a sample of cells. However, such techniquesmay be too complicated to perform on individual cells in large samples.In such methods, RNA transcripts are generally purified from lysed cells(i.e., cells that have been broken apart), followed by conversion of theRNA transcripts into complementary DNA (cDNA) using reversetranscription. The cDNA sequences can then be sequenced using NGS. Insuch a procedure, all of the cDNA sequences are mixed together beforesequencing, such that RNA expression is measured for a whole sample andindividual sequences cannot be linked back to an individual cell.

Methods for uniquely labeling or barcoding transcripts from individualcells can involve the manual separation of individual cells intoseparate reaction vessels and can require specialized equipment. Analternative approach to sequencing individual transcripts in cells is touse microscopy to identify individual fluorescent bases. However, thistechnique can be difficult to implement and limited to sequencing a lownumber of cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments disclosed herein will become more fully apparent fromthe following description and appended claims, taken in conjunction withthe accompanying drawings.

FIG. 1 depicts ligation of nucleic acid tags to form a label or barcode.

FIG. 2 is a schematic representation of the formation of cDNA by in situreverse transcription. Panel A depicts a cell that is fixed andpermeabilized. Panel B depicts addition of a poly(T) primer, which cantemplate the reverse transcription of polyadenylated transcripts. PanelC depicts addition of a random hexamer, which can template the reversetranscription of substantially any transcript. Panel D depicts theaddition of a primer that is designed to target a specific transcriptsuch that only a subset of transcripts may be amplified. Panel E depictsthe cell of Panel A after reverse transcription, illustrating a cDNAhybridized to an RNA.

FIG. 3A depicts non-templated ligation of a single-stranded adapter toan RNA fragment.

FIG. 3B depicts ligation of a single-stranded adapter using a partialduplex with random hexamer primers.

FIG. 4 depicts primer binding.

FIG. 5 depicts primer binding followed by reverse transcription.

FIG. 6 depicts DNA-tagged antibodies for use in labeling cellularproteins.

FIG. 7 depicts aptamers for use in labeling cellular proteins.

FIG. 8 is a schematic representation of the dividing, tagging, andpooling of cells, according to an embodiment of the present disclosure.As depicted, cells can be divided between a plurality of reactionvessels. One cell is highlighted to show its path through theillustrated process.

FIG. 9A depicts an exemplary workflow, according to an embodiment of thepresent disclosure.

FIG. 9B depicts an exemplary workflow, according to another embodimentof the present disclosure.

FIG. 10 depicts a reverse transcription primer (BC_0055), according toan embodiment of the present disclosure.

FIG. 11 depicts an annealed, first-round barcode oligo, according to anembodiment of the present disclosure.

FIG. 12 depicts an annealed, second-round barcode oligo, according to anembodiment of the present disclosure.

FIG. 13 depicts an annealed, third-round barcode oligo, according to anembodiment of the present disclosure.

FIG. 14 depicts ligation stop oligos, according to an embodiment of thepresent disclosure.

FIG. 15 depicts a single-stranded DNA adapter oligo (BC_0047) ligated tothe 3′ end of a cDNA, according to an embodiment of the presentdisclosure.

FIG. 16 depicts a PCR product formed using primers BC_0051 and BC_0062and the 3′ adapter oligo (BC_0047) after it has been ligated to barcodedcDNA.

FIG. 17 depicts BC_0027, which includes the flow cell binding sequenceand the binding site for the TRUSEQ™ read 1 primer and BC_0063, whichincludes the flow cell binding sequence and the TruSeq multiplex read 2and index binding sequence. FIG. 17 also illustrates a region for asample index, which is GATCTG in this embodiment.

FIG. 18 is a scatter plot, wherein for each unique barcode combinationthe number of reads aligning to the human genome (x-axis) and the mousegenome (y-axis) are plotted.

FIG. 19 illustrates the size of cDNA prior to and after tagmentation.

FIG. 20 illustrates an experiment wherein over 150,000 nuclei from P2and P11 mouse brains and spinal cords were profiled in a singleexperiment employing over six million barcode combinations. By recordingwhich of the four starting samples (P2 spine, P2 brain, P11 spine, orP11 spine) were added to each well, the first-round barcode sequencescan be used to identify which cell/nuclei originated from which sample.

FIGS. 21A-21C show the number of nuclei in each well during three roundsof barcoding. Despite pipetting cells by hand, most wells containapproximately equal numbers of nuclei. Dissociation of the P2 spinalcord resulted in fewer cells than the other samples, explaining thelower number of nuclei in the corresponding first round wells.

FIG. 22 depicts the distribution of P2/P11 brain and spinal cordsingle-nucleus transcriptomes within the oligodendrocyte lineage. Eachnuclei's sample (P2 spine, P2 brain, P11 spine, or P11 spine) can bedetermined by examining the first-round barcode from each cell/nucleibarcode combination.

FIGS. 23A-23D depict an overview of a protocol as provided herein.

FIG. 24 depicts a molecular diagram of a protocol as provided herein.

DETAILED DESCRIPTION

The present disclosure relates generally to methods of uniquely labelingor barcoding molecules within a nucleus, a plurality of nuclei, a cell,a plurality of cells, and/or a tissue. The present disclosure alsorelates to kits for uniquely labeling or barcoding molecules within anucleus, a plurality of nuclei, a cell, a plurality of cells, and/or atissue. The molecules to be labeled may include, but are not limited to,RNAs, cDNAs, DNAs, proteins, peptides, and/or antigens.

It will be readily understood that the embodiments, as generallydescribed herein, are exemplary. The following more detailed descriptionof various embodiments is not intended to limit the scope of the presentdisclosure, but is merely representative of various embodiments.Moreover, the order of the steps or actions of the methods disclosedherein may be changed by those skilled in the art without departing fromthe scope of the present disclosure. In other words, unless a specificorder of steps or actions is required for proper operation of theembodiment, the order or use of specific steps or actions may bemodified.

The term “binding” is used broadly throughout this disclosure to referto any form of attaching or coupling two or more components, entities,or objects. For example, two or more components may be bound to eachother via chemical bonds, covalent bonds, ionic bonds, hydrogen bonds,electrostatic forces, Watson-Crick hybridization, etc.

One aspect of the disclosure relates to methods of labeling nucleicacids. In some embodiments, the methods may comprise labeling nucleicacids in a first cell. The methods may comprise: (a) generatingcomplementary DNAs (cDNAs) within a plurality of cells comprising thefirst cell by reverse transcribing RNAs using a reverse transcriptionprimer comprising a 5′ overhang sequence; (b) dividing the plurality ofcells into a number (n) of aliquots; (c) providing a plurality ofnucleic acid tags to each of the n aliquots, wherein each labelingsequence of the plurality of nucleic acid tags provided into a givenaliquot is the same, and wherein a different labeling sequence isprovided into each of the n aliquots; (d) binding at least one of thecDNAs in each of the n aliquots to the nucleic acid tags; (e) combiningthe n aliquots; and (f) repeating steps (b), (c), (d), and (e) with thecombined aliquot. In various embodiments, the plurality of cells may beselected from eukaryotic cells and prokaryotic cells. In various otherembodiments, the plurality of cells may be selected from, but notlimited to, at least one of mammalian cells, yeast cells, and/orbacterial cells.

In certain embodiments, each nucleic acid tag may comprise a firststrand including a 3′ hybridization sequence extending from a 3′ end ofa labeling sequence and a 5′ hybridization sequence extending from a 5′end of the labeling sequence. Each nucleic acid tag may also comprise asecond strand including an overhang sequence. The overhang sequence mayinclude (i) a first portion complementary to at least one of the 5′hybridization sequence and the 5′ overhang sequence and (ii) a secondportion complementary to the 3′ hybridization sequence. In someembodiments, the nucleic acid tag (e.g., the final nucleic acid tag) maycomprise a capture agent such as, but not limited to, a 5′ biotin. AcDNA labeled with a 5′ biotin-comprising nucleic acid tag may allow orpermit the attachment or coupling of the cDNA to a streptavidin-coatedmagnetic bead. In some other embodiments, a plurality of beads may becoated with a capture strand (i.e., a nucleic acid sequence) that isconfigured to hybridize to a final sequence overhang of a barcode. Inyet some other embodiments, cDNA may be purified or isolated by use of acommercially available kit (e.g., an RNEASY™ kit).

In various embodiments, step (f) (i.e., steps (b), (c), (d), and (e))may be repeated a number of times sufficient to generate a unique seriesof labeling sequences for the cDNAs in the first cell. Stated anotherway, step (f) may be repeated a number of times such that the cDNAs inthe first cell may have a first unique series of labeling sequences, thecDNAs in a second cell may have a second unique series of labelingsequences, the cDNAs in a third cell may have a third unique series oflabeling sequences, and so on. The methods of the present disclosure mayprovide for the labeling of cDNA sequences from single cells with uniquebarcodes, wherein the unique barcodes may identify or aid in identifyingthe cell from which the cDNA originated. In other words, a portion, amajority, or substantially all of the cDNA from a single cell may havethe same barcode, and that barcode may not be repeated in cDNAoriginating from one or more other cells in a sample (e.g., from asecond cell, a third cell, a fourth cell, etc.).

In some embodiments, barcoded cDNA can be mixed together and sequenced(e.g., using NGS), such that data can be gathered regarding RNAexpression at the level of a single cell. For example, certainembodiments of the methods of the present disclosure may be useful inassessing, analyzing, or studying the transcriptome (i.e., the differentRNA species transcribed from the genome of a given cell) of one or moreindividual cells.

As discussed above, an aliquot or group of cells can be separated intodifferent reaction vessels or containers and a first set of nucleic acidtags can be added to the plurality of cDNA transcripts. Vessels orcontainers can also be referred to herein as receptacles, samples, andwells. Accordingly, the terms vessel, container, receptacle, sample, andwell may be used interchangeably herein. The aliquots of cells can thenbe regrouped, mixed, and separated again and a second set of nucleicacid tags can be added to the first set of nucleic acid tags. In variousembodiments, the same nucleic acid tag may be added to more than onealiquot of cells in a single or given round of labeling. However, afterrepeated rounds of separating, tagging, and repooling, the cDNAs of eachcell may be bound to a unique combination or sequence of nucleic acidtags that form a barcode. In some embodiments, cells in a single samplemay be separated into a number of different reaction vessels. Forexample, the number of reaction vessels may include four 1.5 mlmicrocentrifuge tubes, a plurality of wells of a 96-well plate, oranother suitable number and type of reaction vessels.

In certain embodiments, step (f) (i.e., steps (b), (c), (d), and (e))may be repeated a number of times wherein the number of times isselected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, etc. In certain other embodiments, step (f) may berepeated a sufficient number of times such that the cDNAs of each cellwould be likely to be bound to a unique barcode. The number of times maybe selected to provide a greater than 50% likelihood, greater than 90%likelihood, greater than 95% likelihood, greater than 99% likelihood, orsome other probability that the cDNAs in each cell are bound to a uniquebarcode. In yet other embodiments, step (f) may be repeated some othersuitable number of times.

In some embodiments, the methods of labeling nucleic acids in the firstcell may comprise fixing the plurality of cells prior to step (a). Forexample, components of a cell may be fixed or cross-linked such that thecomponents are immobilized or held in place. The plurality of cells maybe fixed using formaldehyde in phosphate buffered saline (PBS). Theplurality of cells may be fixed, for example, in about 1-4% formaldehydein PBS. In various embodiments, the plurality of cells may be fixedusing methanol (e.g., 100% methanol) at about −20° C. or at about 25° C.In various other embodiments, the plurality of cells may be fixed usingmethanol (e.g., 100% methanol), at between about −20° C. and about 25°C. In yet various other embodiments, the plurality of cells may be fixedusing ethanol (e.g., about 70-100% ethanol) at about −20° C. or at roomtemperature. In yet various other embodiments, the plurality of cellsmay be fixed using ethanol (e.g., about 70-100% ethanol) at betweenabout −20° C. and room temperature. In still various other embodiments,the plurality of cells may be fixed using acetic acid, for example, atabout −20° C. In still various other embodiments, the plurality of cellsmay be fixed using acetone, for example, at about −20° C. Other suitablemethods of fixing the plurality of cells are also within the scope ofthis disclosure.

In certain embodiments, the methods of labeling nucleic acids in thefirst cell may comprise permeabilizing the plurality of cells prior tostep (a). For example, holes or openings may be formed in outermembranes of the plurality of cells. TRITON™ X-100 may be added to theplurality of cells, followed by the optional addition of HCl to form theone or more holes. About 0.2% TRITON™ X-100 may be added to theplurality of cells, for example, followed by the addition of about 0.1 NHCl. In certain other embodiments, the plurality of cells may bepermeabilized using ethanol (e.g., about 70% ethanol), methanol (e.g.,about 100% methanol), Tween 20 (e.g., about 0.2% Tween 20), and/or NP-40(e.g., about 0.1% NP-40). In various embodiments, the methods oflabeling nucleic acids in the first cell may comprise fixing andpermeabilizing the plurality of cells prior to step (a).

In some embodiments, the cells may be adherent cells (e.g., adherentmammalian cells). Fixing, permeabilizing, and/or reverse transcriptionmay be conducted or performed on adherent cells (e.g., on cells that areadhered to a plate). For example, adherent cells may be fixed,permeabilized, and/or undergo reverse transcription followed bytrypsinization to detach the cells from a surface. Alternatively, theadherent cells may be detached prior to the separation and/or taggingsteps. In some other embodiments, the adherent cells may be trypsinizedprior to the fixing and/or permeabilizing steps.

In some embodiments, the methods of labeling nucleic acids in the firstcell may comprise ligating at least two of the nucleic acid tags thatare bound to the cDNAs. Ligation may be conducted before or after thelysing and/or the cDNA purification steps. Ligation can comprisecovalently linking the 5′ phosphate sequences on the nucleic acid tagsto the 3′ end of an adjacent strand or nucleic acid tag such thatindividual tags are formed into a continuous, or substantiallycontinuous, barcode sequence that is bound to the 3′ end of the cDNAsequence. In various embodiments, a double-stranded DNA or RNA ligasemay be used with an additional linker strand that is configured to holda nucleic acid tag together with an adjacent nucleic acid in a “nicked”double-stranded conformation. The double-stranded DNA or RNA ligase canthen be used to seal the “nick.” In various other embodiments, asingle-stranded DNA or RNA ligase may be used without an additionallinker. In certain embodiments, the ligation may be performed within theplurality of cells

FIG. 1 illustrates ligation of a plurality of nucleic acid tags to forma substantially continuous label or barcode. For example, after aplurality of nucleic acid tag additions, each cDNA transcript may bebound or linked to a series of nucleic acid tags. Use of a ligase mayligate or covalently link a portion of the nucleic acid tags to form asubstantially continuous label or barcode that is bound or attached to acDNA transcript.

In certain other embodiments, the methods may comprise lysing theplurality of cells (i.e., breaking down the cell structure) to releasethe cDNAs from within the plurality of cells, for example, after step(f). In some embodiments, the plurality of cells may be lysed in a lysissolution (e.g., 10 mM Tris-HCl (pH 7.9), 50 mM EDTA (pH 7.9), 0.2 MNaCl, 2.2% SDS, 0.5 mg/ml ANTI-RNase (a protein ribonuclease inhibitor;AMBION®) and 1000 mg/ml proteinase K (AMBION®)), for example, at about55° C. for about 1-3 hours with shaking (e.g., vigorous shaking). Insome other embodiments, the plurality of cells may be lysed usingultrasonication and/or by being passed through an 18-25 gauge syringeneedle at least once. In yet some other embodiments, the plurality ofcells may be lysed by being heated to about 70-90° C. For example, theplurality of cells may be lysed by being heated to about 70-90° C. forabout one or more hours. The cDNAs may then be isolated from the lysedcells. In some embodiments, RNase H may be added to the cDNA to removeRNA. The methods may further comprise ligating at least two of thenucleic acid tags that are bound to the released cDNAs. In some otherembodiments, the methods of labeling nucleic acids in the first cell maycomprise ligating at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, etc. of thenucleic acid tags that are bound to the cDNAs.

In various embodiments, the methods of labeling nucleic acids in thefirst cell may comprise removing one or more unbound nucleic acid tags(e.g., washing the plurality of cells). For example, the methods maycomprise removing a portion, a majority, or substantially all of theunbound nucleic acid tags. Unbound nucleic acid tags may be removed suchthat further rounds of the disclosed methods are not contaminated withone or more unbound nucleic acid tags from a previous round of a givenmethod. In some embodiments, unbound nucleic acid tags may be removedvia centrifugation. For example, the plurality of cells can becentrifuged such that a pellet of cells is formed at the bottom of acentrifuge tube. The supernatant (i.e., liquid containing the unboundnucleic acid tags) can be removed from the centrifuged cells. The cellsmay then be resuspended in a buffer (e.g., a fresh buffer that is freeor substantially free of unbound nucleic acid tags). In another example,the plurality of cells may be coupled or linked to magnetic beads thatare coated with an antibody that is configured to bind the cell ornuclear membrane. The plurality of cells can then be pelleted using amagnet to draw them to one side of the reaction vessel. In some otherembodiments, the plurality of cells may be placed in a cell strainer(e.g., a PLURISTRAINER® cell strainer) and washed with a wash buffer.For example, the plurality of cells may remain in the cell strainerwhile the wash buffer passes through the cell strainer. Wash buffer mayinclude a surfactant, a detergent, and/or about 5-60% formamide.

As discussed above, the plurality of cells can be repooled and themethod can be repeated any number of times, adding more tags to thecDNAs creating a set of nucleic acid tags that can act as a barcode. Asmore and more rounds are added, the number of paths that a cell can takeincreases and consequently the number of possible barcodes that can becreated also increases. Given enough rounds and divisions, the number ofpossible barcodes will be much higher than the number of cells,resulting in each cell likely having a unique barcode. For example, ifthe division took place in a 96-well plate, after 4 divisions therewould be 964=84,934,656 possible barcodes.

In some embodiments, the reverse transcription primer may be configuredto reverse transcribe all, or substantially all, RNA in a cell (e.g., arandom hexamer with a 5′ overhang). In some other embodiments, thereverse transcription primer may be configured to reverse transcribe RNAhaving a poly(A) tail (e.g., a poly(dT) primer, such as a dT(15) primer,with a 5′ overhang). In yet some other embodiments, the reversetranscription primer may be configured to reverse transcribepredetermined RNAs (e.g., a transcript-specific primer). For example,the reverse transcription primer may be configured to barcode specifictranscripts such that fewer transcripts may be profiled per cell, butsuch that each of the transcripts may be profiled over a greater numberof cells.

FIG. 2 illustrates the formation of cDNA by in situ reversetranscription. Panel A depicts a cell that is fixed and permeabilized.Panel B depicts addition of a poly(T) primer, as discussed above, whichcan template the reverse transcription of polyadenylated transcripts.Panel C depicts addition of a random hexamer, as discussed above, whichcan template the reverse transcription of substantially any transcript.Panel D depicts the addition of a primer that is designed to target aspecific transcript, as discussed above, such that only a subset oftranscripts may be amplified. Panel E depicts the cell of Panel A afterreverse transcription, illustrating a cDNA hybridized to an RNA.

Reverse transcription may be conducted or performed on the plurality ofcells. In certain embodiments, reverse transcription may be conducted ona fixed and/or permeabilized plurality of cells. In some embodiments,variants of M-MuLV reverse transcriptase may be used in the reversetranscription. Any suitable method of reverse transcription is withinthe scope of this disclosure. For example, a reverse transcription mixmay include a reverse transcription primer including a 5′ overhang andthe reverse transcription primer may be configured to initiate reversetranscription and/or to act as a binding sequence for nucleic acid tags.In some other embodiments, a portion of a reverse transcription primerthat is configured to bind to RNA and/or initiate reverse transcriptionmay comprise one or more of the following: a random hexamer, a septamer,an octomer, a nonamer, a decamer, a poly(T) stretch of nucleotides,and/or one or more gene specific primers.

Another aspect of the disclosure relates to methods of uniquely labelingmolecules within a cell or within a plurality of cells. In someembodiments, the method may comprise: (a) binding an adapter sequence,or universal adapter, to molecules within the plurality of cells; (b)dividing the plurality of cells into at least two primary aliquots,wherein the at least two primary aliquots comprise at least a firstprimary aliquot and a second primary aliquot; (c) providing primarynucleic acid tags to the at least two primary aliquots, wherein theprimary nucleic acid tags provided to the first primary aliquot aredifferent from the primary nucleic acid tags provided to the secondprimary aliquot; (d) binding the adapter sequences within each of the atleast two primary aliquots with the provided primary nucleic acid tags;(e) combining the at least two primary aliquots; (f) dividing thecombined primary aliquots into at least two secondary aliquots, the atleast two secondary aliquots comprising at least a first secondaryaliquot and a second secondary aliquot; (g) providing secondary nucleicacid tags to the at least two secondary aliquots, wherein the secondarynucleic acid tags provided to the first secondary aliquot are differentfrom the secondary nucleic acid tags provided to the second secondaryaliquot; and (h) binding the molecules within each of the at least twosecondary aliquots with the provided secondary nucleic acid tags.

In certain embodiments, the method may further comprise step (i), i.e.,repeating steps (e), (f), (g), and (h) with subsequent aliquots. Step(i) can be repeated a number of times sufficient to generate a uniqueseries of nucleic acid tags for the molecules in a single cell. Invarious embodiments, the number of times may be selected from 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,etc. In certain other embodiments, step (i) may be repeated anothersuitable number of times.

In some embodiments, the molecules may be disposed within the cell orwithin the plurality of cells. In some other embodiments, the moleculesmay be coupled to the cell or to the plurality of cells. For example,the molecules may be cell-surface molecules. In yet some otherembodiments, the molecules may be disposed within and/or coupled to thecell or the plurality of cells.

As discussed above, the method may comprise fixing and/or permeabilizingthe plurality of cells prior to step (a). In various embodiments, eachof the nucleic acid tags may comprise a first strand. The first strandmay comprise a barcode sequence including a 3′ end and a 5′ end. Thefirst strand may further comprise a 3′ hybridization sequence and a 5′hybridization sequence flanking the 3′ end and the 5′ end of the barcodesequence, respectively. In some embodiments, each of the nucleic acidtags may comprise a second strand. The second strand may comprise afirst portion complementary to at least one of the 5′ hybridizationsequence and the adapter sequence and a second portion complementary tothe 3′ hybridization sequence.

In certain embodiments, the molecules are macromolecules. In variousembodiments, the molecules are selected from at least one of RNA, cDNA,DNA, protein, peptides, and/or antigens.

In some embodiments, the molecules are RNA and the adapter sequence maybe single-stranded. Furthermore, step (a) may comprise one of ligating a5′ end of the single-stranded adapter sequence to a 3′ end of the RNAand/or ligating a 3′ end of the single-stranded adapter sequence to a 5′end of the RNA. In some other embodiments, the molecules are RNA andstep (a) may comprise hybridizing the adapter sequence to the RNA.

Methods related to binding or coupling an adapter sequence to an RNA canbe used, for example, in RNA transcriptome sequencing, ribosomeprofiling, small RNA sequencing, non-coding RNA sequencing, and/or RNAstructure profiling. In some embodiments, the plurality of cells may befixed and/or permeabilized. The 5′ end of a single-stranded adaptersequence may be ligated to the 3′ end of an RNA (see FIGS. 3A and 3B).In certain embodiments, the ligation may be conducted or performed by T4RNA Ligase 1. In certain other embodiments, the ligation may beconducted by T4 RNA Ligase 1 with a single-stranded adapter sequenceincluding a 5′ phosphate. In various embodiments, the ligation may beconducted by THERMOSTABLE 5′ APPDNA/RNA LIGASE™ (NEW ENGLAND BIOLABS®).In various other embodiments, the ligation may be conducted byTHERMOSTABLE 5′ APPDNA/RNA LIGASE™ with a 5′ pre-adenylatedsingle-stranded adapter sequence. Other suitable ligases and adaptersequences are also within the scope of this disclosure.

In some embodiments, the RNA can be labeled with adapter sequence usinghybridization, for example, via Watson-Crick base-pairing (see FIG. 4 ).After the labeling steps and/or cell lysis, as discussed above, theadapter sequence may be configured to prime reverse transcription toform or generate cDNA (see FIG. 5 ).

The 3′ end of a single-stranded adapter sequence may be ligated to the5′ end of an RNA. In certain embodiments, the ligation may be conductedor performed by T4 RNA Ligase 1. In certain other embodiments, theligation may be conducted by T4 RNA Ligase 1 with an RNA including a 5′phosphate. In various embodiments, the ligation may be conducted byTHERMOSTABLE 5′ APPDNA/RNA LIGASE™ (NEW ENGLAND BIOLABS®). In variousother embodiments, the ligation may be conducted by THERMOSTABLE 5′APPDNA/RNA LIGASE™ with a 5′ pre-adenylated RNA. As stated above, othersuitable ligases and adapter sequences are also within the scope of thisdisclosure.

In some embodiments, the molecules may be cDNA. Methods related tobinding or coupling an adapter sequence to a cDNA can be used, forexample, in RNA transcriptome sequencing. In certain embodiments, theplurality of cells may be fixed and/or permeabilized. Reversetranscription may be performed on the plurality of fixed and/orpermeabilized cells with a primer that includes the adapter sequence onthe 5′ end. As discussed above, the 3′ end of the primer may begene-specific, a random hexamer, or a poly(T) sequence. The resultingcDNA may include the adapter sequence on its 5′ end (see FIG. 5 ).

In some embodiments, wherein the molecules are DNA (e.g., genomic DNA),the method may further comprise digesting the DNA with a restrictionenzyme prior to step (a). Furthermore, step (a) may comprise ligatingthe adapter sequence to the digested DNA.

Methods related to binding or coupling an adapter sequence to a DNA maybe used, for example, in whole genome sequencing, targeted genomesequencing, DNase-Seq, ChIP-sequencing, and/or ATAC-seq. In certainembodiments, one or more restriction enzymes may be used to digest DNAinto at least one of blunt end fragments and/or fragments havingoverhang sequences. A partial double-stranded sequence with thesingle-stranded universal adapter or adapter sequence protruding on oneend can be ligated to the digested genomic DNA. For example, a partialdouble-stranded sequence with the single-stranded adapter sequencehaving an overhang, wherein the overhang is compatible with the overhanggenerated by the one or more restriction enzymes, may be ligated to thedigested genomic DNA.

In various embodiments, adapter sequences can be integrated (e.g.,directly integrated) into genomic DNA using Tn5 transposase and thetransposase can be released to expose the adapter sequences by additionof sodium dodecyl sulfate (SDS). Other transposases and methods ofintegrating the adapter sequences into genomic DNA are also within thescope of this disclosure.

In certain embodiments, the molecules are protein, peptide, and/orantigen, and the adapter sequence may be bound to a unique identifiersequence (e.g., comprising nucleic acids) that is coupled to anantibody. The unique identifier sequence may be configured to uniquelyidentify the antibody to which the unique identifier sequence is bound.Furthermore, step (a) may comprise binding the antibodies, whichcomprise each of the adapter sequence and the unique identifiersequence, to the protein, peptide, and/or antigen. In certain otherembodiments, the molecules are protein, peptide, and/or antigen, and theadapter sequence may be integrated in an aptamer. Furthermore, step (a)may comprise binding the aptamer to the protein, peptide, and/orantigen.

Methods related to binding or coupling an adapter sequence to a protein,a peptide, and/or an antigen may be used, for example, in proteinquantification, peptide quantification, and/or antigen quantification.In various embodiments, the adapter sequence can be attached (e.g.,chemically attached) to an antibody. For example, the adapter sequencecan be attached to an antibody using chemistry known to the skilledartisan for mediating DNA-protein bonds. Antibodies for differentproteins can be labeled with nucleic acid sequences or strands thatinclude a unique identifier sequence in addition to the adaptersequence. The antibody, or set of antibodies, may then be used in animmunostaining experiment to label a protein, or set of proteins, infixed and/or permeabilized cells or tissue (see FIG. 6 ). Subsequently,the cells may undergo a labeling or barcoding procedure as disclosedherein.

In some embodiments, the nucleic acid sequences (e.g., the DNAmolecules) attached or bound to the antibodies can be released from theantibodies and/or adapter sequences. A sequencing reaction can reveal aunique identifier sequence associated with a given protein as well asthe label or barcode associated with a unique cell or cells. In certainembodiments, such a method may reveal or identify the number and/or typeof proteins present in one or more cells.

In various embodiments, a DNA aptamer and/or an RNA aptamer can be usedinstead of, or in addition to, a nucleic acid-modified (or DNA-modified)antibody as described above (see FIG. 7 ). The adapter sequence (andtarget protein-specific antibody) may be integrated (e.g., directlyintegrated) into the sequence of a given aptamer.

Another aspect of the disclosure relates to methods of barcoding nucleicacids within a cell. In some embodiments, the methods of barcodingnucleic acids within a cell may comprise: (a) generating cDNAs within aplurality of cells by reverse transcribing RNAs using a reversetranscription primer comprising a 5′ overhang sequence; (b) dividing theplurality of cells into at least two aliquots; (c) providing a pluralityof nucleic acid tags to each of the at least two aliquots, wherein eachbarcode sequence of the plurality of nucleic acid tags introduced into agiven aliquot is the same, and wherein a different barcode sequence isintroduced into each aliquot; (d) binding at least one of the cDNAs ineach of the at least two aliquots to the nucleic acid tags; (e)combining the at least two aliquots; and (f) repeating steps (b), (c),(d), and (e) at least once with the combined aliquot.

In certain embodiments, each nucleic acid tag may comprise a firststrand comprising a 3′ hybridization sequence extending from a 3′ end ofa barcode sequence and a 5′ hybridization sequence extending from a 5′end of the barcode sequence. Each nucleic acid tag may also comprise asecond strand comprising an overhang sequence, wherein the overhangsequence comprises (i) a first portion complementary to at least one ofthe 5′ hybridization sequence and the 5′ overhang sequence and (ii) asecond portion complementary to the 3′ hybridization sequence.

FIG. 8 depicts dividing, tagging, and pooling of cells, according to anembodiment of the present disclosure. Cells that have been reversetranscribed can be divided between reaction vessels or wells. In FIG. 8, 4 wells are shown. As discussed above, however, any suitable number ofreaction vessels or wells may be used. One cell is highlighted to showits path through the process. As depicted, the highlighted cell firstends up in well ‘a’, wherein it is the 1^(st) tag added to it thathybridizes to the overhang of all the cDNA transcripts (shown in thebox). The tag carries a unique barcode region ‘a’, identifying the wellthat the cell was in. After hybridization, all cells are washed toremove excess tags, regrouped, and then split again between the samenumber of wells. The highlighted cell then ends up in well ‘c’ and has a2^(nd) tag added to it identifying the well it was in. After the secondround, the cells could have taken 4²=16 possible paths through thetubes. The process can be repeated, adding more tags to the cDNAtranscripts and increasing the number of possible paths the cells cantake. FIGS. 9A and 9B depict two exemplary workflows, according toembodiments of the present disclosure.

Another aspect of the disclosure relates to kits for labeling nucleicacids within at least a first cell. In some embodiments, the kit maycomprise at least one reverse transcription primer comprising a 5′overhang sequence. The kit may also comprise a plurality of firstnucleic acid tags. Each first nucleic acid tag may comprise a firststrand. The first strand may include a 3′ hybridization sequenceextending from a 3′ end of a first labeling sequence and a 5′hybridization sequence extending from a 5′ end of the first labelingsequence. Each first nucleic acid tag may further comprise a secondstrand. The second strand may include an overhang sequence, wherein theoverhang sequence may comprise (i) a first portion complementary to atleast one of the 5′ hybridization sequence and the 5′ overhang sequenceof the reverse transcription primer and (ii) a second portioncomplementary to the 3′ hybridization sequence.

The kit may further comprise a plurality of second nucleic acid tags.Each second nucleic acid tag may comprise a first strand. The firststrand may include a 3′ hybridization sequence extending from a 3′ endof a second labeling sequence and a 5′ hybridization sequence extendingfrom a 5′ end of the second labeling sequence. Each second nucleic acidtag may further comprise a second strand. The second strand may comprisean overhang sequence, wherein the overhang sequence may comprise (i) afirst portion complementary to at least one of the 5′ hybridizationsequence and the 5′ overhang sequence of the reverse transcriptionprimer and (ii) a second portion complementary to the 3′ hybridizationsequence. In some embodiments, the first labeling sequence may bedifferent from the second labeling sequence.

In some embodiments, the kit may also comprise one or more additionalpluralities of nucleic acid tags. Each nucleic acid tag of the one ormore additional pluralities of nucleic acid tags may comprise a firststrand. The first strand may include a 3′ hybridization sequenceextending from a 3′ end of a labeling sequence and a 5′ hybridizationsequence extending from a 5′ end of the labeling sequence. Each nucleicacid tag of the one or more additional pluralities of nucleic acid tagsmay also comprise a second strand. The second strand may include anoverhang sequence, wherein the overhang sequence comprises (i) a firstportion complementary to at least one of the 5′ hybridization sequenceand the 5′ overhang sequence of the reverse transcription primer and(ii) a second portion complementary to the 3′ hybridization sequence. Insome embodiments, the labeling sequence may be different in each givenadditional plurality of nucleic acid tags.

In various embodiments, the kit may further comprise at least one of areverse transcriptase, a fixation agent, a permeabilization agent, aligation agent, and/or a lysis agent.

Another aspect of the disclosure relates to kits for labeling moleculeswithin at least a first cell. For example, the kits as disclosed abovemay be adapted to label one or more of RNA, cDNA, DNA, protein,peptides, or antigens within at least a first cell.

Another aspect of the disclosure relates to methods of uniquely labelingRNA molecules within a plurality of cells. The methods may include: (a)fixing and permeabilizing a first plurality of cells prior to step (b),wherein the first plurality of cells are fixed and permeabilized atbelow about 8° C.; (b) reverse transcribing the RNA molecules within thefirst plurality of cells to form complementary DNA (cDNA) moleculeswithin the first plurality of cells, wherein reverse transcribing theRNA molecules includes coupling primers to the RNA molecules, whereinthe primers include at least one of a poly(T) sequence or a randomsequence; (c) dividing the first plurality of cells including cDNAmolecules into at least two primary aliquots, the at least two primaryaliquots including a first primary aliquot and a second primary aliquot;(d) providing primary nucleic acid tags to the at least two primaryaliquots, wherein the primary nucleic acid tags provided to the firstprimary aliquot are different from the primary nucleic acid tagsprovided to the second primary aliquot; (e) coupling the cDNA moleculeswithin each of the at least two primary aliquots with the providedprimary nucleic acid tags; (f) combining the at least two primaryaliquots; (g) dividing the combined primary aliquots into at least twosecondary aliquots, the at least two secondary aliquots including afirst secondary aliquot and a second secondary aliquot; (h) providingsecondary nucleic acid tags to the at least two secondary aliquots,wherein the secondary nucleic acid tags provided to the first secondaryaliquot are different from the secondary nucleic acid tags provided tothe second secondary aliquot; (i) coupling the cDNA molecules withineach of the at least two secondary aliquots with the provided secondarynucleic acid tags; (j) repeating steps (f), (g), (h), and (i) withsubsequent aliquots, wherein the final nucleic acid tags include acapture agent; (k) combining final aliquots; (1) lysing the firstplurality of cells to release the cDNA molecules from within the firstplurality of cells to form a lysate; and/or (m) adding a proteaseinhibitor and/or a binding agent to the lysate such that the cDNAmolecules bind the binding agent.

The method may further include dividing the combined final aliquots intoat least two final aliquots, the at least two final aliquots including afirst final aliquot and a second final aliquot. In some embodiments, thefirst plurality of cells may be fixed and permeabilized at below about8° C., below about 7° C., below about 6° C., below about 5° C., at about4° C., below about 4° C., below about 3° C., below about 2° C., belowabout 1° C., or at another suitable temperature. In certain embodiments,the methods may include splitting the cells. For example, following thelast or final round of barcoding (via ligation), the cells can be pooledbefore lysis and then the cells can be split into different lysatealiquots. Each lysate aliquot may include a predetermined number ofcells.

With reference, for example, to step (m), the protease inhibitor mayinclude phenylmethanesulfonyl fluoride (PMSF),4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), acombination thereof, and/or another suitable protease inhibitor. Withreference, for example, to steps (j), (k), (l), and/or (m), the captureagent may include biotin or another suitable capture agent. Furthermore,the binding agent may include avidin (e.g., streptavidin) or anothersuitable binding agent.

In certain embodiments, the methods of uniquely labeling RNA moleculeswithin a plurality of cells may further include (e.g., after step (m)):(n) conducting a template switch of the cDNA molecules bound to thebinding agent using a template switch oligonucleotide; (o) amplifyingthe cDNA molecules to form an amplified cDNA molecule solution; and/or(p) introducing a solid phase reversible immobilization (SPRI) beadsolution to the amplified cDNA molecule solution to removepolynucleotides of less than about 200 base pairs, less than about 175base pairs, or less than about 150 base pairs (see DeAngelis, M M, etal. Nucleic Acids Research (1995) 23(22):4742). In other words, the cDNAmolecules can be bound to streptavidin beads within a lysate. Templateswitching of the cDNA molecules attached to the beads can be performed(e.g., to add an adapter to the 3′-end of the cDNA molecules). PCRamplification of the cDNA molecules can then be performed, followed bythe addition of SPRI beads to remove polynucleotides of less than about200 base pairs. The ratio of SPRI bead solution to amplified cDNAmolecule solution may be between about 0.9:1 and about 0.7:1, betweenabout 0.875:1 and about 0.775:1, between about 0.85:1 and about 0.75:1,between about 0.825:1 and about 0.725:1, about 0.8:1, or anothersuitable ratio. Furthermore, the SPRI bead solution may include betweenabout 1 M and 4 M NaCl, between about 2 M and 3 M NaCl, between about2.25 M and 2.75 M NaCl, about 2.5 M NaCl, or another suitable amount ofNaCl. The SPRI bead solution may also include between about 15% w/v and25% w/v polyethylene glycol (PEG), wherein the molecular weight of thePEG is between about 7,000 g/mol and 9,000 g/mol (PEG 8000). In variousembodiments, the SPRI bead solution may include between about 17% w/vand 23% w/v PEG 8000, between about 18% w/v and 22% w/v PEG 8000,between about 19% w/v and 21% w/v PEG 8000, about 20% w/v PEG 8000, oranother suitable % w/v PEG 8000.

The methods of uniquely labeling RNA molecules within a plurality ofcells may further include adding a common adapter sequence to the 3′-endof the released cDNA molecules. The common adapter sequence can be anadapter sequence that is the same, or substantially the same, for eachof the cDNA molecules (i.e., within a given experiment). The addition ofthe common adapter may be conducted or performed in a solution includingup to about 10% w/v of PEG, wherein the molecular weight of the PEG isbetween about 7,000 g/mol and 9,000 g/mol. In certain embodiments, thecommon adapter sequence may be added to the 3′-end of the released cDNAmolecules by template switching (see Picelli, S, et al. Nature Methods10, 1096-1098 (2013)).

The step (j) may be repeated a number of times sufficient to generate aunique series of nucleic acid tags for the nucleic acids in a singlecell. For example, the number of times can be selected from 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100.

In various embodiments, the primers of step (b) may further include afirst specific barcode. Stated another way, the first barcode added tothe cDNA molecules in a specific container, mixture, reaction,receptacle, sample, well, or vessel may be predetermined (e.g., specificto the given container, mixture, reaction, receptacle, sample, well, orvessel). For example, 48 sets of different well-specific RT primers maybe used (e.g., in a 48-well plate). Accordingly, if there are 48 samples(e.g., cells, tissues, etc.), each sample can get a unique well-specificbarcode. However, if there are only four samples, each sample can have12 different sets of well-specific RT primers. A user can know which 12correspond to each sample, so the user can recover sample identities.Other numbers of first specific barcodes (or well-specific RT primers)are also within the scope of this disclosure. Such a configuration mayallow or provide for the multiplexing of the method as explained furtherin Example 16.

The methods may further include: (q) reverse transcribing RNA moleculeswithin a second plurality of cells to form cDNA molecules within thesecond plurality of cells, wherein reverse transcribing the RNAmolecules includes coupling specific primers to the RNA molecules,wherein the primers include a second specific barcode and at least oneof a poly(T) sequence or a random sequence, wherein the first specificbarcode is different from the second specific barcode such that the cDNAmolecules from the first plurality of cells can be identified incomparison to the cDNA molecules from the second plurality of cells; (r)dividing the second plurality of cells including cDNA molecules into atleast two primary aliquots, the at least two primary aliquots includinga first primary aliquot and a second primary aliquot; (s) providingprimary nucleic acid tags to the at least two primary aliquots, whereinthe primary nucleic acid tags provided to the first primary aliquot aredifferent from the primary nucleic acid tags provided to the secondprimary aliquot; (t) coupling the cDNA molecules within each of the atleast two primary aliquots with the provided primary nucleic acid tags;(u) combining the at least two primary aliquots; (v) dividing thecombined primary aliquots into at least two secondary aliquots, the atleast two secondary aliquots including a first secondary aliquot and asecond secondary aliquot; (w) providing secondary nucleic acid tags tothe at least two secondary aliquots, wherein the secondary nucleic acidtags provided to the first secondary aliquot are different from thesecondary nucleic acid tags provided to the second secondary aliquot;(x) coupling the cDNA molecules within each of the at least twosecondary aliquots with the provided secondary nucleic acid tags; and/or(y) repeating steps (u), (v), (w), and (x) with subsequent aliquots,wherein the final nucleic acid tags include a capture agent. The stepsabove (e.g., steps (k), (1), and (m)) as used with the first pluralityof cells can also be adapted for use with the second plurality of cells.

In various embodiments, each of the nucleic acid tags may include afirst strand, wherein the first strand includes (i) a barcode sequenceincluding a 3′ end and a 5′ end and (ii) a 3′ hybridization sequence anda 5′ hybridization sequence flanking the 3′ end and the 5′ end of thebarcode sequence, respectively. Each of the nucleic acid tags may alsoinclude a second strand, wherein the second strand includes (i) a firstportion complementary to at least one of the 5′ hybridization sequenceand the adapter sequence and (ii) a second portion complementary to the3′ hybridization sequence.

The methods of uniquely labeling RNA molecules within a plurality ofcells may further include ligating at least two (or more) of the nucleicacid tags that are bound to the cDNA molecules. The ligation may beperformed within the first plurality of cells.

The methods may further include removing unbound nucleic acid tags. Insome embodiments, the methods may include ligating at least two of thenucleic acid tags that are bound to the released cDNA molecules. Themajority of the nucleic acid tag-bound cDNA molecules from a single cellmay include the same series of bound nucleic acid tags. In variousembodiments, the pluralities of cells (e.g., the first and secondpluralities of cells) may be selected from at least one of mammaliancells, yeast cells, and bacterial cells.

Another aspect of the disclosure is directed to methods of labelingnucleic acids within a first cell. In certain embodiments, the methodsmay include: (a) generating cDNA molecules within a plurality of cellsincluding the first cell by reverse transcribing RNAs using at least oneof (i) a first reverse transcription primer including a 5′ overhangsequence, wherein the first reverse transcription primer is configuredto reverse transcribe RNA having a poly(A) tail and/or (ii) a secondreverse transcription primer including a 5′ overhang sequence and atleast one of a random hexamer, a random septamer, a random octomer, arandom nonamer, and a random decamer; (b) dividing the plurality ofcells into a number (n) of aliquots; (c) providing a plurality ofnucleic acid tags to each of the n aliquots; (d) binding at least one ofthe cDNA molecules in each of the n aliquots to the nucleic acid tags;(e) combining the n aliquots; (f) repeating steps (b), (c), (d), and (e)with the combined aliquot; (g) combining final aliquots; (h) lysing theplurality of cells including the first cell to release the cDNAmolecules from within the plurality of cells including the first cell toform a lysate; and/or (i) adding a protease inhibitor and/or a bindingagent to the lysate such that the cDNA molecules bind the binding agent.

With reference, for example, to step (c), each nucleic acid tag mayinclude a first strand including (i) a 3′ hybridization sequenceextending from a 3′ end of a labeling sequence and (ii) a 5′hybridization sequence extending from a 5′ end of the labeling sequence.Each nucleic acid tag may also include a second strand including anoverhang sequence, the overhang sequence including (i) a first portioncomplementary to at least one of the 5′ hybridization sequence and the5′ overhang sequence and (ii) a second portion complementary to the 3′hybridization sequence. In some embodiments, the labeling sequence ofthe plurality of nucleic acid tags provided into a given aliquot may bethe same and a different labeling sequence may be provided into each ofthe n aliquots.

In certain embodiments, step (f) may be repeated a number of timessufficient to generate a unique series of labeling sequences for thecDNA molecules in the first cell. For example, the number of times maybe selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, and 100.

The cDNA molecules may be formed or generated in an aliquot (e.g., areaction mixture). The concentration of the first reverse transcriptionprimer in the aliquot may be between about 0.5 μM and about 10 μM,between about 1 μM and about 7 μM, between about 1.5 μM and about 4 μM,between about 2 μM and about 3 μM, about 2.5 μM, or another suitableconcentration. The concentration of the second reverse transcriptionprimer in the aliquot may be between about 0.5 μM and about 10 μM,between about 1 μM and about 7 μM, between about 1.5 μM and about 4 μM,between about 2 μM and about 3 μM, about 2.5 μM, or another suitableconcentration.

In some embodiments, the methods may include fixing the plurality ofcells prior to step (a). The plurality of cells may be fixed at belowabout 8° C., below about 7° C., below about 6° C., below about 5° C., atabout 4° C., below about 4° C., below about 3° C., below about 2° C.,below about 1° C., or at another suitable temperature. In certainembodiments, the methods may include permeabilizing the plurality ofcells prior to step (a). The plurality of cells may be permeabilized atbelow about 8° C., below about 7° C., below about 6° C., below about 5°C., at about 4° C., below about 4° C., below about 3° C., below about 2°C., below about 1° C., or at another suitable temperature.

The methods of labeling nucleic acids within a first cell may alsoinclude ligating at least two of the nucleic acid tags that are bound tothe cDNA molecules. In various embodiments, the ligation may beperformed within the plurality of cells. The methods may includeremoving unbound nucleic acid tags. Furthermore, at least one of thefirst and second reverse transcription primers may reverse transcribepredetermined RNAs or be configured to reverse transcribe predeterminedRNAs.

In various embodiments, the final nucleic acid tags may include acapture agent. Furthermore, the methods may include lysing the pluralityof cells to release the cDNA molecules from within the plurality ofcells after step (f) to form a lysate. The methods may also includeadding a protease inhibitor and/or a binding agent to the lysate toisolate the cDNA molecules. As discussed above, the protease inhibitormay include PMSF, AEBSF, a combination thereof, and/or another suitableprotease inhibitor. The capture agent may include biotin or anothersuitable capture agent and the binding agent may include avidin (e.g.streptavidin) or another suitable binding agent.

The methods of labeling nucleic acids within a first cell may alsoinclude: (j) conducting a template switch of the cDNA molecules bound tothe binding agent; (k) amplifying the cDNA molecules to form anamplified cDNA molecule solution; and (l) introducing an SPRI beadsolution to the amplified cDNA molecule solution to removepolynucleotides of less than about 200 base pairs, less than about 175base pairs, or less than about 150 base pairs. The ratio of SPRI beadsolution to amplified cDNA molecule solution may be between about 0.9:1and about 0.7:1, between about 0.875:1 and about 0.775:1, between about0.85:1 and about 0.75:1, between about 0.825:1 and about 0.725:1, about0.8:1, or another suitable ratio.

Furthermore, the SPRI bead solution may include between about 1 M and 4M NaCl, between about 2 M and 3 M NaCl, between about 2.25 M and 2.75 MNaCl, about 2.5 M NaCl, or another suitable amount of NaCl. The SPRIbead solution may also include between about 15% w/v and 25% w/v PEG,wherein the molecular weight of the PEG is between about 7,000 g/mol and9,000 g/mol. In various embodiments, the SPRI bead solution may includebetween about 17% w/v and 23% w/v PEG 8000, between about 18% w/v and22% w/v PEG 8000, between about 19% w/v and 21% w/v PEG 8000, about 20%w/v PEG 8000, or another suitable % w/v PEG 8000.

The methods of uniquely labeling RNA molecules within a plurality ofcells may further include adding a common adapter sequence to the 3′-endof the released cDNA molecules. As discussed above, the addition of thecommon adapter may be conducted or performed in a solution comprising upto about 10% w/v of PEG, wherein the molecular weight of the PEG isbetween about 7,000 g/mol and 9,000 g/mol. In certain embodiments, thecommon adapter sequence may be added to the 3′-end of the released cDNAmolecules by template switching.

In certain embodiments, any of the methods described above can beadapted for labeling nucleic acid molecules within a nucleus orplurality of nuclei. For example, the methods may include uniquelylabeling RNA molecules within a plurality of nuclei or labeling nucleicacids within a first nucleus.

Another aspect of the disclosure is directed to kits for labelingnucleic acids within a first cell. The kit may include a first reversetranscription primer including a 5′ overhang sequence and may beconfigured to reverse transcribe RNA having a poly(A) tail. The kit mayalso include a second reverse transcription primer including a 5′overhang sequence and at least one of a random hexamer, a randomseptamer, a random octomer, a random nonamer, and/or a random decamer.

In some embodiments, the kit can include a plurality of first nucleicacid tags. As discussed above, each first nucleic acid tag may include afirst strand including (i) a 3′ hybridization sequence extending from a3′ end of a first labeling sequence and (ii) a 5′ hybridization sequenceextending from a 5′ end of the first labeling sequence. Each firstnucleic acid tag may also include a second strand including an overhangsequence. The overhang sequence may include (i) a first portioncomplementary to at least one of the 5′ hybridization sequence and the5′ overhang sequence of the first and second reverse transcriptionprimers and (ii) a second portion complementary to the 3′ hybridizationsequence.

In certain embodiments, the kit can also include a plurality of secondnucleic acid tags Each second nucleic acid tag may include a firststrand including (i) a 3′ hybridization sequence extending from a3′ endof a second labeling sequence and (ii) a 5′ hybridization sequenceextending from a 5′ end of the second labeling sequence. Each secondnucleic acid tag may also include a second strand including an overhangsequence. The overhang sequence may include (i) a first portioncomplementary to at least one of the 5′ hybridization sequence and the5′ overhang sequence of the first and second reverse transcriptionprimers and (ii) a second portion complementary to the 3′ hybridizationsequence. Furthermore, the first labeling sequence may be different fromthe second labeling sequence.

In various embodiments, the kit can also include a plurality of finalnucleic acid tags. Each final nucleic acid tag may include a firststrand including (i) a 3′ hybridization sequence extending from a 3′ endof a final labeling sequence and (ii) a 5′ hybridization sequenceextending from a 5′ end of the final labeling sequence. Each finalnucleic acid tag may also include a second strand including an overhangsequence. The overhang sequence may include (i) a first portioncomplementary to at least one of the 5′ hybridization sequence and the5′ overhang sequence of the first and second reverse transcriptionprimers and (ii) a second portion complementary to the 3′ hybridizationsequence. Each final nucleic acid tag may also include a capture agent.Furthermore, the final labeling sequence may be different from the firstand second labeling sequences (and/or any other labeling sequences). Insome embodiments, the kit may also include at least one of a reversetranscriptase, a fixation agent, a permeabilization agent, a ligationagent, a lysis agent, a protease inhibitor, and/or any other suitablecomponent.

As will be understood by one of ordinary skill in the art, eachembodiment disclosed herein can comprise, consist essentially of, orconsist of its particular stated element, step, ingredient, orcomponent. As used herein, the transition term “comprise” or “comprises”means includes, but is not limited to, and allows for the inclusion ofunspecified elements, steps, ingredients, or components, even in majoramounts. The transitional phrase “consisting of” excludes any element,step, ingredient or component not specified. The transition phrase“consisting essentially of” limits the scope of the embodiment to thespecified elements, steps, ingredients or components, and to those thatdo not materially affect the embodiment.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent disclosure. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques. When further clarity is required, the term “about”has the meaning reasonably ascribed to it by a person skilled in the artwhen used in conjunction with a stated numerical value or range, i.e.,denoting somewhat more or somewhat less than the stated value or range,to within a range of ±20% of the stated value; ±19% of the stated value;±18% of the stated value; ±17% of the stated value; ±16% of the statedvalue; ±15% of the stated value; ±14% of the stated value; ±13% of thestated value; ±12% of the stated value; ±11% of the stated value; ±10%of the stated value; ±9% of the stated value; ±8% of the stated value;±7% of the stated value; ±6% of the stated value; ±5% of the statedvalue; ±4% of the stated value; ±3% of the stated value; ±2% of thestated value; or ±1% of the stated value.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the disclosure (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the disclosure and does not pose alimitation on the scope of the disclosure otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the disclosure.

Groupings of alternative elements or embodiments of the disclosuredisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Definitions and explanations used in the present disclosure are meantand intended to be controlling in any future construction unless clearlyand unambiguously modified in the following examples or when applicationof the meaning renders any construction meaningless or essentiallymeaningless in cases where the construction of the term would render itmeaningless or essentially meaningless, the definition should be takenfrom Webster's Dictionary, 3rd Edition or a dictionary known to those ofordinary skill in the art, such as the Oxford Dictionary of Biochemistryand Molecular Biology (Ed. Anthony Smith, Oxford University Press,Oxford, 2004).

EXAMPLES

The following examples are illustrative of disclosed methods andcompositions. In light of this disclosure, those of skill in the artwill recognize that variations of these examples and other examples ofthe disclosed methods and compositions would be possible without undueexperimentation.

Example 1—Fixation and Reverse Transcription

NIH/3T3 (mouse) and Hela-S3 (human) cells can be grown to confluence ontwo separate 10 cm cell culture plates. The cells can be rinsed twicewith 10 ml 1× phosphate buffered saline (PBS), 1 ml of 0.05% trypsin canbe added to each plate, and the plates can be incubated at 37° C. for 5minutes. The cells can be detached by tilting each plate at a 45° anglewhile pipetting trypsin across the plates, which can be continued untilall, or substantially all, of the cells are detached. Each cell line canbe transferred into its own 15 ml conical centrifuge tube (FALCON™) 2 mlof Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum(FBS) can be added to each tube. The number of cells in each tube can becalculated (e.g., with a hemocytometer or on a flow cytometer). Forexample, 200 μl of the sample can be transferred from each tube intoseparate 1.7 ml microcentrifuge tubes (EPPENDORF®) and 100 μl of thesample can be run on an ACCURI™ Flow Cytometer to calculate the cellconcentration.

The same number of cells from each tube can be combined into a newsingle 15 ml conical centrifuge tube (FALCON™), using as many cells aspossible. A 5 minute spin can be conducted at 500×g in a 15 ml conicalcentrifuge tube (FALCON™). It may be helpful to use a bucket centrifugeso that the cells are pelleted at the bottom of the tube rather than onthe side of the tube. The liquid can be aspirated without disturbing thecell pellet and the cells can be resuspended in 500 μl of 4%formaldehyde. The cells can then be left at room temperature (i.e.,20-25° C.) for 10 minutes. 1.5 ml of 0.5% TRITON™ X-100 can be added tothe tube and mixed gently with a pipette. The tube can them be spun at500×g for 5 minutes. Again, the liquid can be aspirated withoutdisturbing the pellet and the pellet can be washed twice with 1 ml PBSwithout resuspending the pellet. If washing disturbs the pellet, thesecond wash can be skipped. The pellet can then be resuspended in 1 ml0.1 N HCl and incubated at room temperature for 5 minutes.

2 ml of Tris-HCl (pH 8.0) can be added to a new 15 ml conical centrifugetube (FALCON™). The fixed cells in HCl, from above, can be transferredto the tube with Tris-HCl so as to neutralize the HCl. The number ofcells in the tube can then be calculated as discussed above (e.g., witha hemocytometer or on a flow cytometer). The fixed cells in Tris-HCl canbe spun down at 500×g for 5 minutes and the liquid can be aspiratedwithout disturbing the pellet. The pellet can be washed twice with 1 mlRNase-free molecular grade water, without disturbing the pellet. Thecells can then be resuspended to a concentration of 2.5 million cells/ml(to do this, the concentration calculated before the last spin step canbe used).

A reverse transcription mix can be made (55 μl M-MuLV reversetranscriptase buffer (ENZYMATICS®), 55 μl M-MuLV reverse transcriptase(ENZYMATICS®), 5.5 μl dNTPs (25 mM per base), 3.44 μl RNase inhibitor(ENZYMATICS®, 40 units/μl), 210.4 μl nuclease-free water, and 2.75 μl RTPrimer (BC_0055, 100 μM)). In a well of a 24-well cell culture plate,300 μl of the reverse transcription mix can be combined with 200 μl ofthe fixed cells (˜500,000 cells) and mixed gently by pipetting. Themixture can then be incubated at room temperature for 10 minutes toallow the reverse transcription primer to anneal and the mixture canthen be incubated at 37° C. in a humidified incubator overnight (i.e.,˜16 hours).

A primer that can be used for reverse transcription (BC_0055) isdepicted in FIG. 10 . This is an anchored primer, designed to bind thestart of a poly(A) tail of a messenger RNA. The primer may besynthesized with all 4 bases at the 3′ end (N) and every base except Tat the second-most 3′ position (V). The primer can also include 15consecutive dTs. In some embodiments, the primer may include more than15 dTs. In some other embodiments, the primer may include fewer than 15dTs. In embodiments wherein the primer includes fewer than 15 dTs, themelting temperature of the primer may be lowered. The domain s0 may nothybridize to messenger RNAs, but may instead provide an accessiblebinding domain for a linker oligo. The primer also includes a 5′phosphate that can allow ligation of the primer to another oligo by T4DNA ligase.

Example 2—Preparation of Barcodes

The barcodes were ordered in 96-well plates at 100 μM concentrations.Each barcode was annealed with its corresponding linker oligo (see FIGS.10-12 ).

FIG. 11 depicts an annealed, first-round barcode oligo. 96 first-roundbarcode oligos with unique sequences in domain i8a were used. In thefirst round, the unique sequence in domain i8a is the region of thesequence that is used as a barcode. By varying 8 nucleotides, there are65,536 possible unique sequences. In some embodiments, more than 8nucleotides may be present in domain i8a. In some other embodiments,fewer than 8 nucleotides may be present in domain i8a. The first-roundbarcodes were preannealed to a linker strand (BC_0056) throughcomplementary sequences in domain s1. The linker strand can includecomplementary sequence to part of the reverse transcription primer(domain s0) that can allow it to hybridize and bring the 3′ end of thefirst-round barcodes in close proximity to the 5′ end of the reversetranscription primer. The phosphate of the reverse transcription primercan then be ligated to the 3′ end of the first-round barcodes by T4 DNAligase. The domain s2 can provide an accessible binding domain for alinker oligo to be used in another round of barcoding. The first-roundbarcode oligos can include a 5′ phosphate that can allow ligation to the3′ end of another oligo by T4 DNA ligase.

FIG. 12 depicts an annealed, second-round barcode oligo. 96 second-roundbarcode oligos with unique sequences in domain i8b were used. In thesecond round, the unique sequence in domain i8b is the region of thesequence that is used as a barcode. By varying 8 nucleotides, there are65,536 possible unique sequences. In some embodiments, more than 8nucleotides may be present in domain i8b. In some embodiments, less than8 nucleotides may be present in domain i8b. The second-round barcodescan be preannealed to a linker strand (BC_0058) through complementarysequences in domain s3. The linker strand can include complementarysequence to part of the first-round barcode oligo (domain s2) that canallow it to hybridize and bring the 3′ end of the first-round barcodesin close proximity to the 5′ end of the second-round barcode oligo. Thephosphate of the first-round barcode oligo can then be ligated to the 3′end of the second-round barcodes by T4 DNA ligase. The domain s4 canprovide an accessible binding domain for a linker oligo to be used inanother round of barcoding. The second-round barcode oligos can includea 5′ phosphate that can allow ligation to the 3′ end of another oligo byT4 DNA ligase.

FIG. 13 depicts an annealed, third-round barcode oligo. 96 third-roundbarcode oligos with unique sequences in domain i8c were used. In thethird round, the unique sequence in domain i8c is the region of thesequence that is used as a barcode. By varying 8 nucleotides, there are65,536 possible unique sequences. In some embodiments, more than 8nucleotides may be present in domain i8c. In some other embodiments,less than 8 nucleotides may be present in domain i8c. The third round ofbarcodes can be preannealed to a linker strand (BC_0060, SEQ ID NO. 16)through complementary sequences in domain s5. The linker strand caninclude complementary sequence to part of the second-round barcode oligo(domain s4) that can allow it to hybridize and bring the 3′ end of thesecond-round barcodes in close proximity to the 5′ end of thethird-round barcode oligo. The phosphate of the second-round barcodeoligo can then be ligated to the 3′ end of the third-round barcodes byT4 DNA ligase. The third-round barcode oligos can be synthesized withunique molecular identifiers (UMI; see Islam, et. al. Nature Methods,2014) consisting of 10 random nucleotides (domain UMI: NNNNNNNNNN). Dueto PCR amplification bias, multiple sequencing reads can originate fromthe cDNA. Using a UMI, each cDNA may be counted only once. Thethird-round barcodes can also include a domain corresponding to part ofthe ILLUMINA® TruSeq adapter. The third-round barcodes can besynthesized with a biotin molecule at the 5′ end so that fully barcodedcDNA can be isolated with streptavidin coated magnetic beads.

Starting from a 100 μM stock of each barcode oligo (i.e., in 96-wellplates, one for each round), 11 μl of barcode oligo were transferred to96-well PCR plates. To the plate with the round 1 barcodes, 9 μl ofBC_0056 (100 μM stock) were added to each well. To the plate with theround 2 barcodes, 9 μl of BC_0058 (100 μM stock) were added to eachwell. To the plate with the round 3 barcodes, 9 μl of BC_0060 (SEQ IDNO. 16) (100 μM stock) were added to each well. Each plate was thenplaced in a thermocycler, with the following program, to anneal thebarcodes with the corresponding linker oligo: heat to 90° C., reduceheat 0.1° C./second, and stop when the temperature reaches 25° C. 2.2 μlwere transferred from each well having the round 1 barcodes into a new96-well plate (referred to as plate L1). 3.8 μl were transferred fromeach well with the round 2 barcodes into a new 96-well plate (referredto as plate L2). 6.1 μl were transferred from each well with the round 3barcodes into a new 96-well plate (referred to as plate L3).

Example 3—Preparation of Ligation Stop Oligos

After each round of ligation, the ligation can be stopped by adding anexcess of oligo that is complementary to the linker strands (see FIG. 14). To stop each barcode ligation, oligo strands that are fullycomplementary to the linker oligos can be added. These oligos can bindthe linker strands attached to unligated barcodes and displace theunligated barcodes through a strand displacement reaction. The unligatedbarcodes can then be completely single-stranded. As T4 DNA ligase isunable to ligate single-stranded DNA to other single-stranded DNA, theligation reaction will stop progressing. To ensure that all linkeroligos are bound by the complementary oligos, a molar excess of thecomplementary oligos (relative to the linker oligos) is added. To stopthe first-round ligation, BC_0064 (complementary to BC_0056) is added.To stop the second-round ligation, BC_0065 (complementary to BC_0058) isadded. To stop the third-round ligation, BC_0066 (SEQ ID NO. 18)(complementary to BC_0060, SEQ ID NO. 16) is added.

Dilutions can be prepared for each stop ligation strand (BC_0064,BC_0065, BC_0066 (SEQ ID NO. 18)) as follows: 264 μl stop ligationstrand (BC_0064, BC_0065, BC_0066 (SEQ ID NO. 18)), 300 μl 10×T4 DNALigase Buffer, and 636 μl nuclease-free water.

Example 4—Ligation of Barcodes to cDNA

5 μl 10% TRITON™ X-100 can be added to the reverse transcriptionreaction (to a final concentration of 0.1%) in the above-described24-well plate. The reverse transcription (RT) reaction with cells can betransferred to a 15 ml conical centrifuge tube (FALCON™). The RTreactions can be spun for 10 minutes at 500×g and resuspended in 2 mlnuclease-free water. The cells can be combined with ligase mix (600 μl10×T4 ligase buffer, 2040 μl of nuclease-free water, all of theresuspended cells (2000 μl), 100 μl of T4 DNA Ligase (NEW ENGLANDBIOLABS®, 400,000 units/ml), and 60 μl of 10% TRITON™ X-100) in adisposable pipetting reservoir (10 ml)). The cells and ligase mix can bemixed by gently tilting the reservoir back and forth several times.Using a multichannel pipette, 40 μl of the cells in the ligase mix canbe added to each well of annealed round 1 barcodes (plate L1). Each wellcan be mixed by pipetting up and down gently 2-3 times. The cells in theligase mix can be incubated at 37° C. for 60 minutes.

10 μl of the diluted BC_0064 can be added to each well to stop theligation. The samples can then be incubated at 37° C. for 30 minutes.All of the cells can be collected in a new disposable pipettingreservoir (10 ml). The cells can be passed through a 40 μM strainer intoa new disposable pipetting reservoir (10 ml) using a 1 ml pipette. 100μl of T4 DNA ligase (NEW ENGLAND BIOLABS®, 400,000 units/ml) can beadded to the cells in reservoir. The cells and ligase mix can be mixedby gently tilting the reservoir back and forth several times and using amultichannel pipette, 40 μl of the cells in the ligase mix can be addedto each well of annealed round 2 barcodes (plate L2). Each well can bemixed by pipetting up and down gently 2-3 times and the samples can thenbe incubated at 37° C. for 60 minutes.

10 μl of the diluted BC_0065 can be added to each well to stop theligation. The samples can be incubated at 37° C. for 30 minutes and thecells can then be collected in a new disposable pipetting reservoir (10ml). The cells can be passed through a 40 μM strainer into a newdisposable pipetting reservoir (10 ml) using a 1 ml pipette. 100 μl ofT4 DNA ligase (NEW ENGLAND BIOLABS®, 400,000 units/ml) can be added tothe cells in the reservoir. The cells and ligase mix can be mixed bygently tilting the reservoir back and forth several times. Using amultichannel pipette, 40 μl of the cells in the ligase mix can be addedto each well of annealed round 3 barcodes (plate L3). Each well can thenbe mixed by pipetting up and down gently 2-3 times and the samples canbe incubated at 37° C. for 60 minutes.

10 μl of the diluted BC_0066 (SEQ ID NO. 18) can be added to each wellto stop the ligation. The samples can be incubated at 37° C. for 30minutes. All the cells can be collected in a new disposable pipettingreservoir (10 ml). The cells can be transferred to a 15 ml conicalcentrifuge tube (FALCON™) and the tube can be filled with wash buffer(nuclease-free water, 0.05% Tween 20, and 25% formamide) to 15 ml. Thesamples can be incubated for 15 minutes at room temperature. The cellscan then be pelleted at 500×g for 10 minutes and the liquid can beremoved without disturbing the pellet. Each tube of cells can beresuspended in 100 μl PBS and the cells can be counted (e.g., on ahemocytometer or on a flow cytometer). In one example, 57,000 cells wereretained. The number of cells to be sequenced can be chosen. In oneexample, the cells were split into 25 cell, 250 cell, 2,500 cell, and25,000 cell aliquots. 300 μl of lysis buffer (10 mM NaF, 1 mM Na₃VO₄,0.5% DOC buffer, and 0.5% TRITON™ X-100) can be added to each of thecell aliquots and each of the cell aliquots can be passed through a 25gauge needle eight times.

Example 5—Binding Barcoded cDNA to Streptavidin Coated Beads

First, DYNABEADS® MYONE™ Streptavidin C1 beads can be resuspended. 20 μlof resuspended DYNABEADS® MYONE™ Streptavidin C1 beads (for each aliquotof cells) can be added to a 1.7 ml microcentrifuge tube (EPPENDORF®).The beads can be washed 3 times with 1× phosphate buffered saline Tween20 (PBST) and resuspended in 20 μl PBST. 900 μl PBST can be added to thecell aliquot and 20 μl of washed C1 beads can be added to the aliquot oflysed cells. The samples can be placed on a gentle roller for 15 minutesat room temperature and then washed 3 times with 800 μl PBST using amagnetic tube rack (EPPENDORF®). The beads can then be resuspended in100 μl PBS.

Example 6—RNase Treatment of Beads

A microcentrifuge tube (EPPENDORF®) comprising a sample can be placedagainst a magnetic tube rack (EPPENDORF®) for 2 minutes and then theliquid can be aspirated. The beads can be resuspended in an RNasereaction (3 μl RNase Mix (ROCHE™), 1 μl RNase H (NEW ENGLAND BIOLABS®),5 μl RNase H 10× Buffer (NEW ENGLAND BIOLABS®), and 41 μl nuclease-freewater). The sample can be incubated at 37° C. for 1 hour, removed from37° C., and placed against a magnetic tube rack (EPPENDORF®) for 2minutes. The sample can be washed with 750 μl of nuclease-freewater+0.01% Tween 20 (H₂O-T), without resuspending the beads and keepingthe tube disposed against the magnetic tube rack. The liquid can then beaspirated. The sample can be washed with 750 μl H₂O-T withoutresuspending the beads and while keeping the tube disposed against themagnetic tube rack. Next, the liquid can be aspirated while keeping thetube disposed against the magnetic tube rack. The tube can then beremoved from the magnetic tube rack and the sample can be resuspended in40 μl of nuclease-free water.

Example 7-3′ Adapter Ligation

With reference to FIG. 15 , to facilitate PCR amplification, asingle-stranded DNA adapter oligo (BC_0047) can be ligated to the 3′ endof cDNA. To prevent concatemers of the adapter oligo, dideoxycytidine(ddC) can be included at the 3′ end of the adapter oligo. BC_0047 wasgenerated with a phosphate at the 5′ end and ddC at the 3′ end. Severalenzymes are capable of ligating single-stranded oligo to the 3′ end ofsingle-stranded DNA. Herein, T4 RNA ligase 1 (NEW ENGLAND BIOLABS®) wasused. Thermostable 5′ AppDNA/RNA Ligase (NEW ENGLAND BIOLABS®) can alsobe used with a preadenylated adaptor oligo.

Specifically, 20 μl of the RNase-treated beads can be added to a singlePCR tube. 80 μl of ligase mix (5 μl T4 RNA Ligase 1 (NEW ENGLANDBIOLABS®), 10 μl 10×T4 RNA ligase buffer, 5 μl BC_0047 oligo at 50 μM,50 μl 50% PEG 8000, and 10 μl 10 mM ATP) can be added to the 20 μl ofbeads in the PCR tube. 50 μl of the ligase mixed with the beads can betransferred into a new PCR tube to prevent too many beads from settlingto the bottom of a single tube and the sample can be incubated at 25° C.for 16 hours.

Example 8—Generating ILLUMINA® Compatible Sequencing Products

Ligation reactions from both PCR tubes can be combined into a single 1.7ml microcentrifuge tube (EPPENDORF®). 750 μl of H₂O-T can be added toeach sample. Each of the tubes can be placed on a magnetic tube rack(EPPENDORF®) for 2 minutes, the liquid can be aspirated, and the samplescan be resuspended in 40 μl water. The samples can be transferred to PCRtubes. 60 μl of PCR mix can be added to each tube (50 μl 2× PHUSION® DNAPolymerase Master Mix (THERMO FISHER™ Scientific), 5 μl BC_0051 (10 μM),and 5 μl BC_0062 (SEQ ID NO. 17) (10 μM)). 10 cycles of PCR can be run(98° C. for 3 minutes, repeat 10 times (98° C. for 10 seconds, 65° C.for 15 seconds, and 72° C. for 60 seconds), and 72° C. for 5 minutes).FIG. 16 depicts the PCR product. After the 3′ adapter oligo (BC_0047)has been ligated to barcoded cDNA, the cDNA can be amplified using PCR.As shown in FIG. 16 , the primers BC_0051 and BC_0062 (SEQ ID NO. 17)were used.

The PCR samples from the previous step can be procured and the magneticbeads can be displaced to the bottom of each tube with a magnet. 90 μlof PCR reaction can be transferred to a new 1.7 ml without transferringany of the magnetic beads. 10 μl of nuclease-free water can be added toeach of the 1.7 ml tubes to a total volume of 100 μl. 60 μl of AMPURE™beads can be added to the 100 μl of PCR reaction (0.6×SPRI) and boundfor 5 minutes. The tubes can be placed against a magnet for 2 minutesand the samples can be washed with 200 μl of 70% ethanol (30 secondwait) without resupending the beads. The samples can be washed againwith 200 μl of 70% ethanol (30 second wait) without resupending thebeads and then the samples can be air dried for 5-10 minutes until theethanol has evaporated.

Each of the samples can be resuspended in 40 μl of nuclease-free water.The tubes can be placed against a magnetic rack for 2 minutes. While themicrocentrifuge tubes (EPPENDORF®) are still disposed against themagnetic rack, 38 μl of solution can be transferred to a new 1.7 mltube, without transferring beads. 62 μl of nuclease-free water can beadded to the samples to a total volume of 100 μl. 60 μl of AMPURE™ beadscan then be added to 100 μl of the PCR reaction (0.6×SPRI) and bound for5 minutes. The tubes can be placed against a magnet for 2 minutes andthen the samples can be washed with 200 μl of 70% ethanol (30 secondwait) without resupending the beads. The samples can be washed againwith 200 μl 70% ethanol (30 second wait) without resupending the beadsand then the samples can be air dried for 5-10 minutes until the ethanolhas evaporated.

The samples can be resuspended in 40 μl of nuclease-free water and eachtube can be placed against a magnetic rack for 2 minutes. While the tubeis still disposed against the magnetic rack, 38 μl of solution to a new1.7 ml tube, without transferring any beads. 20 μl of the 38 μl elutioncan be added to an optical PCR tube. Furthermore, a PCR mix can be addedto the tube (25 μl PHUSION® DNA Polymerase Master Mix (THERMO FISHER™Scientific), 2.5 μl BC_0027 (10 PM), 2.5 μl BC_0063 (10 μM), and 2.5 μl20× EVAGREEN® (BIOTIUM™)). Following the PCR depicted in FIG. 16 , thefull ILLUMINA® adapter sequences can be introduced through another roundof PCR. As depicted in FIG. 17 , BC_0027 includes the flow cell bindingsequence and the binding site for the TRUSEQ™ read 1 primer. BC_0063includes the flow cell binding sequence and the TruSeq multiplex read 2and index binding sequence. There is also a region for the sample index,which is GATCTG in this example.

The above samples can be run on a qPCR machine with the followingcycling conditions: 1) 98° C. for 3 minutes, 2) 98° C. for 10 seconds,3) 65° C. for 15 seconds, 4) 72° C. for 60 seconds, and 5) repeat steps2-4 (e.g., 10-40 times, depending on when fluorescence stops increasingexponentially). The tube can be transferred to a thermocycler set to 72°C. for 5 minutes. The qPCR reaction can be run on a 1.5% agarose gel for40 minutes and a 450-550 bp band can be removed and gel extracted(QIAQUICK® Gel Extraction Kit). The products can be sequenced on anILLUMINA® MISEQ™ using paired end sequencing. The sequencing primers canbe the standard TRUSEQ™ multiplex primers. Read 1 can sequence the cDNAsequence, while read 2 can cover the unique molecular identifier as wellas the 3 barcode sequences (8 nucleotides each). Index read 1 can beused to sequence sample barcodes, so multiple samples may be sequencedtogether.

Example 9—Data Analysis

Sequencing reads were grouped by cell barcodes (three barcodes of eightnucleotides each, 96×96×96=884,736 total combinations). Each barcodecombination should correspond to the cDNA from a single cell. Only readswith valid barcodes were retained. The sequencing reads with eachbarcode combination were aligned to both the human genome and the mousegenome. Reads aligning to both genomes were discarded. Multiple readswith the same unique molecular identifier were counted as a single read.Reads with unique molecular identifiers with two or less mismatches wereassumed to be generated by sequencing errors and were counted as asingle read. For each unique barcode combination the number of readsaligning to the human genome (x-axis) and the mouse genome (y-axis) wereplotted (see FIG. 18 ). As each cell is either mouse or human, it shouldideally include only one type of RNA. So an ideal plot would have everypoint along the x- or y-axis. The fact that most points in the plot ofFIG. 18 are near an axis indicates that the method is viable.

Each point in the plot corresponds to cDNA with the same combination ofbarcodes and should represent the cDNA from a single cell. For eachpoint, the number of reads that map uniquely to the mouse genome areplotted on the y-axis, while the number of reads that map uniquely tothe human genome are plotted on the x-axis. If cDNAs with a specificcombination of barcodes came from a single cell, all of the cDNA withthe specific combination of barcodes should map completely to the humangenome or completely to the mouse genome. As stated above, the fact thatmost barcode combinations map close to either the x-axis (human cells)or the y-axis (mouse cells) indicates that the method can indeed producesingle-cell RNA sequencing data.

Example 10—Methods of Uniquely Labeling Molecules of a Plurality ofCells

For the following disclosed protocol the projected experimental time istwo (2) days. As indicated below, RNase inhibitor may be added to thebuffers. Accordingly, when any buffer includes the term “+RI”, thisindicates that ENZYMATICS® RNase inhibitor should be added to a finalconcentration of 0.1 U/μL. The centrifugation steps may be performedwith a swinging bucket rotor. In some embodiments, using a fixed anglecentrifuge may lead to more cell loss. Depending on the tissue type,centrifugation speeds may need to be changed to optimize cell retention(e.g., smaller cells=higher speeds).

For DNA barcoding plate generation the following may be needed: i) three96-well plates from IDT®, reverse transcription barcode primers,ligation round 1, and ligation round 2 stock DNA oligo plates (100 μM);ii) two linker oligos, BC_0215 (SEQ ID NO. 30) and BC_0060 (SEQ ID NO.16) (Note: these are assumed to be in stock concentration of 1 mM, thus,correct for volume if another stock concentration is to be used (e.g.,100 μM stocks)); and iii) Six 96-well PCR plates (e.g., three (3) stockplates that will last at least 10 experiments, and three (3) plates forfirst experiment). Note that this can generate 100 μL of DNA barcodesfor each well. Each experiment generally requires only 4 μL/well of thereverse transcription primer solution which can last for 25 experiments.Each experiment generally requires only 10 μL/well of the barcode/linkersolutions, so these plates can last for a total of 10 experiments.

Round 1 Reverse Transcription Barcoded Primers (final concentrations of12.5 μM random hexamer and 12.5 μM 15 dT primers in each of 48wells): 1) using a multichannel pipette, add 12.5 μL of rows A-D in theIDT® reverse transcription barcode primers to rows A-D of the BC stock96-well PCR plate; 2) using a multichannel pipette, add 12.5 μL of rowsE-H in the IDT® reverse transcription barcode primers to rows A-D of theBC stock 96-well PCR plate (mixing poly dT with random hexamer primerhere); 3) add 75 μl of water to rows A-D of the BC stock 96-well PCRplate.

Round 2 Ligation Round (final concentrations of 12 μM barcodes, 11 μMlinker-BC_0215 (SEQ ID NO. 28)): 1) using a multichannel pipette, add 12μL of IDT® round 2 barcodes to R1 stock 96-well PCR plate; 2) add 138.6μl of BC_0215 (SEQ ID NO. 28) (1 mM) to 10.9494 mL water in a basin(BC_0215_dil); and 3) using a multichannel pipette, add 88 μLBC_0215_dil to each well of R2 stock 96 well PCR plate.

Ligation Round 3 (final concentrations of 14 μM barcodes, 13 μMlinker-BC_0060, SEQ ID NO. 16): 1) using a multichannel pipette, add 14μL of round 3 barcodes to R3 stock 96-well PCR plate; 2) add 163.8 μl ofBC_0060 (SEQ ID NO. 16) (1 mM) to 10.6722 mL water in a basin(BC_0060_dil); and 3) using a multichannel pipette, add 86 μL BC_0060(SEQ ID NO. 16) to each well R3 stock 96 well PCR plate.

For each ligation plate (R2 and R3, not including reverse transcriptionbarcodes), anneal the barcode and linker oligos with the followingthermocycling protocol: 1) heat to 95° C. for two (2) minutes and 2)ramp down to 20° C. for at a rate of 0.1° C./s; and 3) 4° C.

Aliquot out 10 μL of each barcode/linker stock plate into three (3) new96-well PCR plates. These are the plates that should be used for DNAbarcoding in the split-pool ligation steps in the protocol.

I. Nuclei Extraction (Optional): 1) prepare the following items, a) keepDounce homogenizer at 4° C. until use, b) 15 ml of 1×PBS+37.5SUPERASE-IN™ +19 μl ENZYMATICS® RNase inhibitor (kept on ice), and c)precool centrifuge to 4° C.

2) Make NIM1 buffer (Table 1):

TABLE 1 NIM1 Buffer Stock Final Volume Reagent ConcentrationConcentration (μL) Sucrose 1.5M   250 mM  2,500 KCl 1M 25 mM 375 MgCl₂1M  5 mM 75 Tris buffer, pH 8 1M 10 mM 150 Water NA NA 11,900 FinalVolume 15,000

3) Make the homogenization buffer (Table 2):

TABLE 2 Homogenization Buffer Stock Final Volume Reagent ConcentrationConcentration (μL) NIM1 Buffer 1.5M 4,845 1 mM DTT 1 mM 1 μM 5ENZYMATICS ® RNase- 40 U/μL 0.4 U/μl 50 In (40 U/μl) SUPERASE-IN ™ (2020 U/μL 0.2 U/μl 50 U/μL) 10% TRITON ™ X-100 10% NA 50 Final Volume5,000

4) Dounce homogenizer: a) add tissue/cells sample to Dounce homogenizer;if cells, resuspend in 700 μl of homogenization buffer; b) addhomogenization buffer to ˜700 μl; c) perform 5 strokes of loose pestle;d) perform 10-15 of tight pestle; e) add homogenization buffer up to 1ml; and f) check cell lysis with 5 μl trypan blue and 5 μl cells onhemocytometer to see if nuclei have been released.

5) Filter homogenates with a 40 μm strainer into 5 ml EPPENDORF™ tubes(or 15 mL FALCON™ tubes). Tilting the filter 450 while straining overthe tube can ensure that the lysate passes through as intended. Note:this straining process is different from the straining processes below.

6) Spin for 4 minutes at 600×g (4° C.) and remove supernatant (can leaveabout 20 μL to avoid aspirating pellet). 7) Resuspend in 1 ml of1×PBS+RI. 8) Add 10 μl of BSA. 9) Centrifuge at 600×g for 4 minutes. 10)Resuspend in 200 μl 1×PBS+RI.

11) Take 50 μl of the resuspended cells from step 4 and add 150 μl of1×PBS+RI. Count sample on hemocytometer and/or flow-cytometer. Thevolume of resuspended cells from the step 4 can be changed based on theconsiderations of the user. 12) Pass cells through a 40 μm strainer intoa fresh 15 mL FALCON™ tube and place on ice (see note below on step 4 ofII. Fixation and Permeabilization). 13) Resuspend the desired number ofnuclei (typically 2M) in 1 mL 1×PBS+RI and proceed with step 5 in thefollowing Fixation and Permeabilization protocol.

II. Fixation and Permeabilization: 1) prepare the following buffers(calculated for two experiments): a) a 1.33% formalin (360 μL of 37%formaldehyde solution (SIGMA®)+9.66 ml PBS) solution and store at 4° C.;b) 6 mL of 1×PBS+RI (15 μL of SUPERASE-IN™ and 7.5 μL of ENZYMATICS®RNase inhibitor); c) 2 mL of 0.5×PBS+RI (5 μL of SUPERASE-IN™ and 2.5 μlof ENZYMATICS® RNase inhibitor); d) 500 μL of 5% TRITON™ X-100+RI (2 μLof SUPERASE-IN™); e) 500 μL of 100 mM Tris pH 8.0+2 μL SUPERASE-IN™; andf) set the centrifuge to 4° C.

2) Pellet cells by centrifuging at 500×g for 3 minutes at 4° C. (somecells may require faster centrifugation). 3) Resuspend cells in 1 mL ofcold PBS+RI. Keep cells on ice between these steps. 4) Pass cellsthrough a 40 μm strainer into a fresh 15 mL FALCON™ tube and place onice. Note: the cell resuspension is not likely to passively go throughthe strainer, which can cause cell loss. Instead, with a 1 ml pipettefilled with the resuspension, press the end of the tip directly onto thestrainer and actively push the liquid through. The motion should takeabout one (1) second. 5) Add 3 mL of cold 1.33% formaldehyde (finalconcentration of 1% formaldehyde). Fix cells on ice for 10 minutes. 6)Add 160 μL of 5% TRITON™ X-100+RI to fixed cells and mix by gentlypipetting up and down 5 times with a 1 mL pipette. Permeabilize cellsfor 3 minutes on ice. 7) Centrifuge cells at 500×g for 3 minutes at 4°C. 8) Aspirate carefully and resuspend cells in 500 μL of cold PBS+RI.9) Add 500 μL of cold 100 mM Tris-HCl, pH 8.0. 10) Add 20 μL of 5%TRITON™ X-100. 11) Centrifuge cells at 500×g for 3 minutes at 4° C. 12)Aspirate and resuspend cells in 300 μl of cold 0.5×PBS+RI.

13) Run cells through a 40 μM strainer into a new 1.7 mL tube (see noteabove on step 4 of II. Fixation and Permeabilization). 14) Count cellsusing a hemocytometer or a flow-cytometer and dilute the cell suspensionto 1,000,000 cells/mL. While counting cells, keep cell suspension onice. Note: this step will dictate how many cells enter the split-poolrounds. It will be possible to sequence only a subset of the cells thatenter the split-pool rounds (can be done during sub-library generationat lysis step). The total number of barcode combinations that will beused should be calculated to determine the maximum number of cells thatcan be sequenced with minimal barcode collisions. Without being bound byany one particular theory, the number of cells that will be processedshould not exceed more than 5% of total barcode combinations. Generally,a dilution between 500 k to 1M cells/mL can be used here (equates to 4-8k cells going into each well for reverse transcription barcodingrounds).

III. Reverse Transcription: 1) aliquot out 4 μL of the RT barcodes stockplate into the top four (4) rows (48 wells) of a new 96-well plate.Cover this plate with an adhesive plate seal until ready for use.

2) Create the following reverse transcription (RT) mix on ice (Table 3):

TABLE 3 RT Mix Stock Desired Per Volume in Mix (48 Reagent ConcentrationConcentration Reaction wells + 10%) 5X RT Buffer 5X 1X 4 211.2ENZYMATICS ® 40 u/μL 0.25 u/μL 0.125 6.6 RNase Inhibitor SUPERASE-IN ™20 U/μL 0.25 U/μL 0.25 13.2 RNase Inhibitor dNTPs 10 mM (per base) 500μM 1 52.8 Maxima H Minus 200 u/μL 20 u/μl 2 105.6 Reverse TranscriptaseH₂O NA NA 0.625 33 Total Volume 8 422.4

3) Add 8 μL of the RT mix to each of the top 48 wells. Each well shouldnow contain a volume of 12 μL. 4) Add 8 μL of cells in 0.5×PBS+RI toeach of the top 48 wells. Each well should now contain a volume of 20μL. 5) Add the plate into a thermocycler with the following protocol: a)50° C. for 10 minutes; b) cycle three (3) times, i) 8° C. for 12seconds, ii) 15° C. for 45 seconds, iii) 20° C. for 45 seconds, and iv)30° C. for 30 seconds, v) 42° C. for 2 minutes, vi) 50° C. for 3minutes; c) 50° C. for 5 minutes; and d) 4° C. forever.

6) Place the RT plate on ice. 7) Prepare 2 mL of 1×NEB buffer 3.1 with20 μL of ENZYMATICS® RNase Inhibitor. 8) Transfer each RT reaction to a15 mL FALCON™ tube (also on ice). 9) Add 9.6 μL of 10% TRITON™ X-100 toget a final concentration of 0.1%. 10) Centrifuge pooled RT reaction for3 minutes at 500×g. 11) Aspirate supernatant and resuspend into 2 mL of1×NEB buffer 3.1+20 μL ENZYMATICS® RNase Inhibitor.

IV. Ligation Barcoding: 1) make the following ligation master mix on ice(Table 4):

TABLE 4 Ligation Master Mix Stock Final Volume Reagent ConcentrationConcentration (μL) Water NA NA 1337.5 T4 Ligase Buffer 10X 10X 1X 500ENZYMATICS ® RNase 40 U/μL 0.32 U/μL 40 Inhibitor SUPERASE-IN ™ 20 U/μL0.05 U/μL 12.5 BSA 20 mg/mL 0.2 mg/mL 50 T4 DNA Ligase 400 U/μL 8 U/μL100 Total Volume 2040Note: final concentration takes added volume of DNA barcodes intoaccount. Concentration of this mix is not the final concentration attime of barcoding.

2) Add the 2 mL of cells in NEB buffer 3.1 into the ligation mix. Themix should now have a volume of 4.04 mL. 3) Add the mix into a basin. 4)Using a multichannel pipet, add 40 μL of ligation mix (with cells) intoeach well of the round 1 DNA barcode plate. 5) Cover the round 1 DNAbarcode plate with an adhesive plate seal and incubate for 30 minutes at37° C. with gentle rotation (50 rpm). 6) Make the round 1 blockingsolution and add it to a new basin (Table 5).

TABLE 5 Round 1 Blocking Solution Stock Final Volume ReagentConcentration Concentration (μL) BC_0216 100 μM 26.4 μM 316.8 10X LigaseBuffer 10X 2.5X 300 Water NA NA 583.2 Final Volume 1200 μL

7) Remove the round 1 DNA barcoding plate from the incubator an removethe cover. 8) Using a multichannel pipet, add 10 μL of the round 1blocking solution to each of the 96 wells in the round 1 DNA barcodingplate. 9) Cover the round 1 DNA barcode plate with an adhesive plateseal and incubate for 30 minutes at 37° C. with gentle rotation (50rpm). 10) Remove round 1 DNA barcoding plate from the incubator, removecover, and pool all cells into a new basin. 11) Pass all the cells fromthis basin through a 40 μm strainer into another basin (see note aboveon step 4 of Fixation and Permeabilization). 12) Add 100 μL of T4 DNAligase to the basin and mix by pipetting about 20 times. 13) Using amultichannel pipette, add 50 μL of cell/ligase solution into each wellof the round 2 DNA barcode plate. 14) Cover the round 2 DNA barcodeplate with an adhesive plate seal and incubate for 30 minutes at 37° C.with gentle rotation (50 rpm).

15) Make the round 2 blocking solution and add it to a new basin (Table6).

TABLE 6 Round 2 Blocking Solution Stock Final Volume ReagentConcentration Concentration (μL) BC_0066 100 μM 11.5 μM 369 EDTA 0.5M125 mM 800 Water NA NA 2031 Final Volume 3200 μL

16) Remove the round 2 DNA barcoding plate from the incubator and removethe cover. 17) Using a multichannel pipet, add 20 μL of the round 2blocking and termination solution to each of the 96 wells in the round 2DNA barcoding plate. 18) Pool all cells into a new basin (no incubationfor the final blocking step). 19) Pass all the cells from this basinthrough a 40 μm strainer into a 15 mL FALCON™ tube (see note above onstep 4 of Fixation and Permeabilization). 19) Count cells on a flowcytometer. Make sure cells are well mixed before aliquoting sample forcounting.

V. Lysis: 1) make the 2× lysis buffer (Table 7).

TABLE 7 2X Lysis Buffer Stock Final Volume Reagent ConcentrationConcentration (2X) (mL) Tris, pH 8.0 1M  20 mM 0.5 NaCl 5M 400 mM 2EDTA, pH 8.0 0.5M   100 mM 5 SDS 10% 4.4% 11 Water NA NA 6.5 FinalVolume 25

2) If white precipitate appears, warm at 37° C. until precipitate isback in solution (about 10-15 minutes).

3) Make the following wash buffer (Table 8).

TABLE 8 Wash Buffer Reagent Volume (μL) 1X PBS 4000 10% TRITON ™ X-10040 SUPERASE-IN ™ RNase Inhibitor 10 Final Volume 4050

4) Add 70 μl of 10% TRITON™ X-100 to the cells (˜0.1% finalconcentration). 5) Centrifuge for 5 minutes at 1000×g in 15 ml tube.Note: the pellet for the steps below can be very small and it may not bevisible. 6) Aspirate supernatant, leave about 30 μl to avoid removingpellet, a) if possible, remove as much supernatant as possible with a 20μL pipet. 7) Resuspend with 4 mL of wash buffer. 8) Centrifuge for 5minutes at 1000×g. 9) Aspirate supernatant and resuspend in 50 μl1×PBS+RI.

10) Dilute 5 μl into 195 μL of 1×PBS and count via flow cytometry. Ortake 5 μl into 5 μl of 1×PBS and count on hemocytometer (it can be hardto distinguish debris from cells). 11) Determine how many sub-librariesto generate (# of sub-libraries=# of tubes needed), and how many cellsto have for each of these sub-libraries. 12) Aliquot the desired numberof cells for each sub-library into new 1.7 mL tubes. Add 1×PBS to eachtube to a final volume of 50 μL.

13) Add 50 μL of 2× Lysis buffer to each tube. 14) Add 10 μL ofProteinase K (20 mg/mL) to each lysate. 15) Incubate at 55° C. for 2hours with shaking at 200 rpm. 16) Stopping point (optional): freezelysate(s) at −80° C.

VI. Prepare Buffers. First make the following stock solutions (Tables9-11).

TABLE 9 Phenylmethanesulfonyl Fluoride (PMSF) 100 mM PMSF

TABLE 10 2X B&W 2X B&W Reagents Volume 1M Tris-HCl pH 8.0 500 μL 5M NaCl20 ml EDTA, 0.5M 100 μl Nuclease Free Water 29.4 ml Total 50 mL

TABLE 11 1X B&W-T 1X B&W-T Reagents Volume 1M Tris-HCl pH 8.0 100 μL 5MNaCl 4 ml EDTA, 0.5M 20 μl Tween 20 10% 100 μl Nuclease Free Water 15.78ml Total 20 mL

Then make the following smaller aliquots (with added RNase inhibitor;Tables 12-14):

TABLE 12 1X B&W-T +RI Volume per Number of Samples (μL) Reagent 1 2 3 45 6 7 8 1X B&W-T 3600.0 4200.0 4800.0 5400.0 6000.0 6600.0 7200.0 7800.0SUPERASE- 1.5 3.0 4.5 6.0 7.5 9.0 10.5 12.0 IN ™ Final 3601.5 4203.04804.5 5406.0 6007.5 6609.0 7210.5 7812.0 Volume

TABLE 13 2X B&W +RI Volume per Number of Samples (μL) Reagent 1 2 3 4 56 7 8 2X B&W 110.0 220.0 330.0 440.0 550.0 660.0 770.0 880.0 SUPERASE-2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 IN ™ Final Volume 112.0 224.0 336.0448.0 560.0 672.0 784.0 896.0

TABLE 14 Tris-T +RI Volume per Number of Samples (μL) Reagent 1 2 3 4 56 7 8 Tris-HCI pH 8.0 600.0 1200.0 1800.0 2400.0 3000.0 3600.0 4200.04800.0 Tween-20 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 (10%) SUPERASE-1.5 3.0 4.5 6.0 7.5 9.0 10.5 12.0 IN ™ Final Volume 607.5 1215.0 1822.52430.0 3037.5 3645.0 4252.5 4860.0

VII. Purification of cDNA. Note: agitation steps have been performed ona vortexer with a foam 1.7 mL tube holder on a low setting (2/10).

Wash MYONE™ C1 DYNABEADS®: 1) for each lysate to be processed, add 44 μLof MYONE™ C1 DYNABEADS® to a 1.5 mL tube (e.g., 1 lysate=44 μL, 2lysates=88 μL, 3 lysates=132 μL, etc.). 2) Add 800 μL of 1× B&W-Tbuffer. 3) Place sample against a magnetic rack and wait until liquidbecomes clear (1-2 minutes). 4) Remove supernatant and resuspend beadsin 800 μL of 1×B&W-T buffer. 5) Repeat steps 3-4 two more times for atotal of 3 washes. 6) Place sample against a magnetic rack and waituntil liquid becomes clear. 7) Resuspend beads in 100 μL (per sample)2×B&W buffer+RI.

Sample Binding to Streptavidin: 1) add 5 μL of 100 μM PMSF to eachsample and leave at room temperature for 10 minutes. 2) Add 100 pal ofresupended C1 beads to each tube. 3) To bind cDNA to C1 beads, agitateat room temperature for 60 minutes. 4) Place sample against a magneticrack and wait until liquid becomes clear (1-2 minutes). 5) Removesupernatant and resuspend beads in 250 μL of 1×B&W. 6) Agitate beads for5 minutes at room temperature. 7) Repeat steps 5 and 6. 8) Removesupernatant and resuspend beads in 250 μL of 10 mM Tris+T. 9) Agitatebeads for 5 minutes at room temperature. 10) Leave beads in final washsolution on ice.

Template Switch. Prepare the following mix depending on the number ofsamples (Table 15).

TABLE 15 Mix Volume per Number of Samples (μL) Reagent 1 2 3 4 5 6 7 8Water 88.0 176.0 264.0 352.0 440.0 528.0 616.0 704.0 Maxima RT 44.0 88.0132.0 176.0 220.0 264.0 308.0 352.0 Buffer FICOLL ® PM-400 44.0 88.0132.0 176.0 220.0 264.0 308.0 352.0 (20%) 10 mM dNTPs (each, total is 4022.0 44.0 66.0 88.0 110.0 132.0 154.0 176.0 mM) RNase Inhibitor 5.5 11.016.5 22.0 27.5 33.0 38.5 44.0 TSO (BC_0127) 5.5 11.0 16.5 22.0 27.5 33.038.5 44.0 Maxima RT RNaseH Minus 11.0 22.0 33.0 44.0 55.0 66.0 77.0 88.0Enzyme Total 220.0 440.0 660.0 880.0 1100.0 1320.0 1540.0 1760.0

1) Place sample against a magnetic rack and wait until liquid becomesclear. 2) With sample still on magnetic rack, remove supernatant andwash with 250 μL of water (do not resuspend beads this time). 3)Resuspend sample in 200 μl of Template Switch Mix. 4) Incubate at roomtemp for 30 minutes with agitation or rolling. 5) Incubate at 42° C. for90 minutes with agitation or rolling (the incubator has been shaken at100 rpm).

6) Potential Stopping Point. If stopping, perform the following(otherwise skip to next section): a) place sample against a magneticrack and wait until liquid becomes clear and b) resuspend in 250 μLTris-T.

VIII. cDNA Amplification. Prepare the following PCR mix depending on thenumber of samples (Table 16).

TABLE 16 PCR Mix Volume per Number of Samples (μL) Reagent 1 2 3 4 5 6 78 KAPA ™ HIFI 121.00 242.00 363.00 484.00 605.00 726.00 847.00 968.00 2XMaster Mix BC_0108 9.68 19.36 29.04 38.72 48.40 58.08 67.76 77.44 (10μM) BC_0062 9.68 19.36 29.04 38.72 48.40 58.08 67.76 77.44 (10 μM) Water101.64 203.28 304.92 406.56 508.20 609.84 711.48 813.12 Total 242.0484.0 726.0 968.0 1210.0 1452.0 1694.0 1936.01) Place sample against a magnetic rack and wait until liquid becomesclear. 2) With sample against magnet wash with 250 μL nuclease-freewater (do not resuspend). 3) Resuspend sample with 220 μL PCR mix andsplit equally into four (4) different PCR tubes.

4) Run the following thermocycling program: a) 95° C., 3 minutes; b) 98°C., 20 seconds; c) 65° C., 45 seconds; d) 72° C., 3 minutes; e) repeat(b-d) four times (5 total cycles); and f) 4° C., hold.

5) Combine all four (4) reactions into a single 1.7 mL tube. Make sureto resuspend any beads that may be stuck to the bottom or sides of thePCR tubes before combining reactions. 6) Place sample against a magneticrack and wait until liquid becomes clear. 7) Transfer 200 μL ofsupernatant to four (4) optical grade qPCR tubes (50 μL in each tube).8) Add 2.5 μL of 20× EVAGREEN® to each qPCR tube. 9) Run the followingqPCR program (make sure to remove samples, once signal starts to leaveexponential phase to prevent over-amplification): a) 95° C., 3 minutes;b) 98° C., 20 seconds; c) 67° C., 20 seconds; d) 72° C., 3 minutes; e)repeat (b-d) until signal plateaus out of exponential amplification; f)72° C., 5 minutes; and g) 4° C., hold.

10) Optional: run an agarose gel or bioanalyze resulting qPCR. Therewill likely be a combination of cDNA and dimer present.

SPRI Size Selection (0.8×). 1) Combine qPCR reactions into a singletube. 2) Take out 180 μL of the pooled qPCR reaction and place in new1.7 mL tube. 3) Add 144 μL of KAPA™ Pure Beads to tube and vortexbriefly to mix. Wait five (5) minutes to bind DNA. 4) Place tube againstmagnetic rack and wait until liquid becomes clear. 5) Remove thesupernatant. 6) With tubes still on magnetic rack, wash with 750 μL 85%ethanol. Do not resuspend beads. 7) Repeat step 6.

8) Remove ethanol and air dry bead (˜5 minutes). Do not let beadsoverdry and crack. 9) Resuspend beads from each tube in 20 μL of water.Once beads are fully resuspended in the water, incubate the tube at 37°C. for 10 minutes. 10) Bind tubes against magnetic rack and wait untilliquid becomes clear. 11) Transfer 18.5 μL of elutant into a new opticalgrade PCR tube. 12) Run a bioanalyzer trace on 10 μL of the elutant.

13) If no dimer is present after size selection, jump directly to“Tagmentation and ILLUMINA® Amplicon Generation” section below. If dimeris still present, proceed to step 14 to perform a second amplificationand size selection step. This may be necessary for cells with low RNAcontent, but should not be necessary for cells with high RNA content(e.g., HeLa-S3, NIH/3T3, etc.).

Second qPCR (Optional). 14) Make the following qPCR mix depending on thenumber of samples (Table 17).

TABLE 17 qPCR Mix Volume per Number of Samples (μL) Reagent 1 2 3 4 5 67 8 KAPA ™ HIFI 27.50 55.00 82.50 110.00 137.50 165.00 192.50 220.00 2XMaster Mix BC_0062 2.20 4.40 6.60 8.80 11.00 13.20 15.40 17.60 (10 μM)BC_0108 2.20 4.40 6.60 8.80 11.00 13.20 15.40 17.60 (10 μM) EVAGREEN ®2.75 5.50 8.25 11.00 13.75 16.50 19.25 22.00 20X Sample 20.35 40.7061.05 81.40 101.75 122.10 142.45 162.80 Total 55.00 110. 00 165.00220.00 275.00 330.00 385.00 440.00

15) Add 31.5 μL of the qPCR master mix to each optical PCR tube with theprevious PCR sample. Gently mix by flicking and spin tubes briefly in atable centrifuge to remove air bubbles. 16) Run the following qPCRprogram (make sure to remove samples, once signal starts to leaveexponential phase to prevent over-amplification): a) 95° C., 3 minutes;b) 98° C., 20 seconds; c) 67° C., 20 seconds; d) 72° C., 3 minutes; e)repeat (b-d) until signal plateaus out of exponential amplification; f)72° C., 5 minutes; and g) 4° C., hold.

17) Run an agarose gel or bioanalyze resulting qPCR. While there maystill be dimer, amplified cDNA should be clearly visible between 500 bpto 2500 bp (see FIG. 19 , left side for expected size distribution).

Second SPRI Size Selection (0.8×). 18) Combine qPCR reactions into asingle tube. 19) Take out 40 μL of the qPCR reaction and place in new1.7 mL tube. 20) Add 32 μL of KAPA™ Pure Beads to each tube and vortexbriefly to mix. Wait 5 minutes to bind DNA.

21) Place tubes against magnetic rack and wait until liquid becomesclear. 22) Remove the supernatant. 23) With tubes still on magneticrack, wash with 750 μL 85% ethanol. 24) Repeat step 5. 25) Removeethanol and air dry bead (˜5 minutes). Do not let beads overdry andcrack. 26) Resuspend beads from each tube in 20 μL of water and wait 5minutes. 27) Bind tubes against magnetic rack and wait until liquidbecomes clear. 28) Transfer 18.5 μL of elutant into a 1.7 mL tube. 29)Run an agarose gel or bioanalyze resulting qPCR. There should be almostno dimer at this point. If dimer remains, perform another round of qPCRfollowed by another 0.8×SPRI size selection.

IX. Tagmentation and ILLUMINA® Amplicon Generation. 1) Quibit amplifiedcDNA and dilute to 0.12 ng/μL. 2) Preheat a thermocycler to 55° C. 3)For each sample, combine 600 pg of purified cDNA with H₂O in a totalvolume of 5 μl. 4) To each tube, add 10 μl of Nextera TD buffer and 5 μlof Amplicon Tagment enzyme (the total volume of the reaction is now 20μl). Mix by pipetting about 5 times and spin down. 5) Incubate at 55° C.for 5 minutes. 6) Add 5 μl of Neutralization Buffer. Mix by pipettingabout 5 times and spin down (bubbles are normal). 7) Incubate at roomtemperature for 5 minutes.

8) Add to each PCR tube in the following order: i) 15 μl of Nextera PCRmix; ii). 8 μl H₂O; iii) 1 μl of 10 μM (N7 indexed primer, one ofBC_0076-BC_0083 (SEQ ID NOS. 19-26)); and iv) 1 μl of 10 μM Nextera(BC_0118, SEQ ID NO. 28) N501 oligo. 9) Run the following thermocyclingprogram: i) 95° C., 30 seconds; ii) 12 cycles of: a) 95° C., 10 seconds;b) 55° C., 30 seconds; and c) 72° C., 30 seconds; and iii) then, 72° C.,5 minutes and 4° C. forever.

10) Transfer 40 μl out of the 50 μL reaction to a 1.7 mL tube. 11) Add28 μL of KAPA™ Pure Beads to do a 0.7× cleanup. Elute in 20 μl. 12)Bioanalyze resulting sample and quibit before sequencing (see FIG. 19 ,right side, for expected size distribution).

X. ILLUMINA® Sequencing. 1) Use a paired-end sequencing run with a 150bp kit. 2) Set read1 to 66 nt (transcript sequence). 3) Set read2 to 94nt (cell-specific barcodes and UMI). 4) Include a 6 nt read 1 index toready sub-library indices.

Example 11—Fixing Cells on Ice and Keeping Cells on Ice forPermeabilization

Unique molecular indices (UMIs) are random molecular barcodes that areadded to each transcript/cDNA prior to amplification. They allow thecomputational removal of PCR duplicates, since these duplicates have thesame UMI sequence. Thus, each original transcript will only be countedonce, even if multiple PCR duplicates are sequenced.

The measure of UMIs per cell indicates how many unique RNA molecules canbe detected per cell, which is directly related to how efficiently themolecules can be barcoded and processed to enable detection by nextgeneration sequencing. This measure is generally the gold standard inthe field and most researchers in the single cell RNA-sequencing spacedetermine how well their experiment worked based on how many UMIs percell are detected with a given amount of raw sequencing reads (e.g.,10,000 UMIs per cell with 50,000 sequencing reads per cell).

For each of Examples 11-16, all conditions within the same table (i.e.,Table 18, Table 19, Table 20, Table 21, and Table 22) were sequenced tothe same saturation level, allowing for an accurate comparison acrossconditions. Each condition had the same number of raw sequencing readscompared to another condition, indicating that differences in UMIsdetected per cell detected were due to change in conditions rather thansequencing depth.

In other protocols (see, e.g., Rosenberg, A B, et al. BioRxiv (2017):105163) formaldehyde fixation is performed at room temperature. Herein,experiments were performed showing a substantial improvement when cellswere kept on ice (e.g., at 4° C.) during fixation and permeabilization(see Table 18).

TABLE 18 Room Temperature vs. 4° C. Unique RNA Molecules Detected PerCell Approach (UMIs per cell) Cells kept at room temperature duringfixation 1002.72 and permeabilization Cells kept at 4° C. duringfixation and 1524.59 permeabilization

Example 12-Adding a Protease Inhibitor (PMSF) and Binding Directly toStreptavidin Beads

In other protocols (see, e.g., Rosenberg, A B, et al. BioRxiv (2017):105163) nucleic acids are first isolated from the lysis solution with anSPRI bead cleanup before binding desired nucleic acids (containing 5′biotin) to streptavidin beads. Herein, it was found that adding PMSF tolysates and then directly adding streptavidin beads (thereby skippingthe first SPRI isolation of nucleic acids) improved the number of uniquemolecules that were detected per cell (see Table 19).

TABLE 19 Streptavidin Selection and PMSF Unique RNA Molecules DetectedPer Cell Approach (UMIs per cell) SPRI cleanup, followed by streptavidin1562.32 selection Streptavidin selection directly in lysis buffer1894.85 (with protease inhibitor PMSF)

Example 13-0.6× vs. 0.8×SPRI Cleanup

In other protocols (see, e.g., Rosenberg, A B, et al. BioRxiv (2017):105163), after cDNA amplification, a 0.6×SPRI cleanup was performed.After cDNA amplification short products (i.e., products under about 200base pairs) can be removed using a SPRI cleanup. Herein, it was foundthat adding a 0.8:1 ratio of SPRI beads to PCR product resulted in moredetected unique RNA molecules per cell than the previous 0.6:1 ratio ofSPRI to PCR products (see Table 20).

TABLE 20 0.6X vs. 0.8X SPRI Cleanup Unique RNA Molecules Detected PerCell Approach (UMIs per cell) 0.6X SPRI cleanup after cDNA amplification1562.32 0.8X SPRI cleanup after cDNA amplification 2553.36

Example 14—Concentrations of Random Hexamer and polydT RT Primers

In other protocols (see, e.g., Rosenberg, A B, et al. BioRxiv (2017):105163) reverse transcription using solely polydT primers for reversetranscription is performed. Herein, it was found that by combiningbarcoded random hexamer primers and altering the concentrations led to asignificant increase in UMIs/cell. With reference to Table 21, Protocol,Version 2.1 uses condition number 4 (729 UMIs/cell). With continuedreference to Table 21, herein, condition number 10 was used (1263UMIs/cell). Accordingly, combining random hexamer and polydT reversetranscription primers together at specific concentrations increasesefficiency of in situ reverse transcription.

TABLE 21 PolydT_15 PolydT_30 Random Hexamer Random Condition micromolar(uM) micromolar (uM) micromolar (uM) PotydT_15 PolydT_30 Hexamer TotalUMIs/cell Number concentration concentration concentration UMIs/cellUMIs/cell UMIs/cell from condition 1 0.5 0 0 245.47 245.47 2 2.5 0 0355.71 355.71 3 5 0 0 549.06 549.06 4 10 0 0 729.03 729.03 5 0 0.5 0452.23 452.23 6 0 2.5 0 344.73 344.73 7 0 5 0 318.44 318.44 8 0 10 0437.13 437.13 9 0.5 0 0.5 621.77 206.27 828.04 10 2.5 0 2.5 618.31644.81 1263.12 11 5 0 5 625.43 445.9 1071.33 12 10 0 10 390.94 383.22774.16

Example 15—Using the First Round of Barcodes as a Sample Identifier

Barcoded reverse transcription primers were used to generate first roundbarcodes in cDNA molecules. These cDNA molecules are then barcodedfurther by appending barcodes to the 5′ end with ligation as describedabove.

A proof of concept is described in Rosenberg, A B, et al. (2018)Science, 360(6385); 176-182, which is hereby incorporated by referencein its entirety. As described therein, an experiment included four (4)unique samples and which cell belonged to which sample was essentiallytracked by observing the first barcode (incorporated through RT). Thissample identification can require knowing the corresponding barcodesequence to each well and which samples were placed into which well.With this information, a lookup table from first round barcode sequencesto sample IDs can be created.

FIGS. 20 and 21 show an experimental setup for multiplexing samples. Inthis specific case, 4 samples were multiplexed over 48 wells in a96-well plate. One can see how this same logic can be expanded tomultiplex up to 96 samples in a single experiment.

With reference to FIG. 22 , analysis of transcriptomes usingT-distributed Stochastic Neighbor Embedding (t-SNE) in an experimentwith four different biological samples is shown. Each point representsthe transcriptome from one individual cell. Using the identity of thebarcodes incorporated during RT (with barcoded RT primers), the sampleidentity of each transcriptome can be determined.

Example 16—Incorporating PEG for Template Switching of DNA/RNA Bound toMagnetic Beads

A biotinylated oligo attached to reverse transcribed RNA (cDNA/RNAduplex) is bound to a streptavidin coated magnetic bead. Then, templateswitching is performed on this cDNA/RNA duplex so that a single commonadapter sequence can incorporated to the 3′ end of cDNA. Incorporatingup to 10% w/v PEG (molecular weight 7000-9000) into the template switchreaction, which includes cDNA/RNA duplex bound to streptavidin coatedmagnetic beads, can improve the efficiency of this step as measured bytranscript detection per cell (Table 22).

TABLE 22 Template Switch with or without PEG 8000 Unique RNA MoleculesDetected Per Cell Approach (UMIs per cell) Template Switch without PEG8000 2645.0 Template Switch with 7.5% PEG 8000 5759.0

Example 18—Exemplary Methods of Uniquely Labeling RNA Molecules

Panel A of FIG. 23 depicts labeling transcriptomes with split-poolbarcoding. In each split-pool round, fixed cells or nuclei can berandomly distributed into wells and transcripts can be labeled withwell-specific barcodes. Barcoded RT primers can be used in the firstround. Second and third round barcodes can be appended to cDNA throughligation. A fourth barcode can be added to cDNA molecules by PCR duringsequencing library preparation. The bottom scheme shows the finalbarcoded cDNA molecule.

Panel B of FIG. 23 shows a species mixing experiment with a libraryprepared from 1,758 whole cells. Human UBCs extend horizontally alongthe x-axis, mouse UBCs are extend vertically along the y-axis, andmixed-species UBCs are disposed between the human and mouse UBCs. Theestimated barcode collision rate is 0.2%, whereas species purity is>99%.

Panel C of FIG. 23 shows UMI counts from mixing experiments performedwith fresh and frozen (stored at −80° C. for 2 weeks) cells and nuclei.Median human UMI counts for fresh cells: 15,365; frozen cells: 15,078;nuclei: 12,113; frozen nuclei: 13,636.

Panel D of FIG. 23 shows measured gene expression by the methodsprovided here is highly correlated between frozen cells and cellsprocessed immediately (Pearson-r: 0.987). Frozen and fresh cells wereprocessed in two different SPLiT-seq experiments.

As illustrated in FIG. 24 , fixed and permeabilized cells can berandomly split into wells that each contain reverse transcriptionprimers with a well-specific barcode. In situ reverse transcriptionconverts RNA to cDNA while appending the well-specific barcode. Cellscan then be pooled and again randomly split into a second set of wells,each containing a unique well-specific barcode. These barcodes can behybridized and ligated to the 5′-end of the barcoded reversetranscription primer to add a second round of barcoding. The cells canbe pooled back together and a subsequent split-ligate-pool round can beperformed. After the last round of ligation, cDNA molecules contain acell-specific combination of barcodes, a unique molecular identifier,and a universal PCR handle on the 5′-end. A fourth barcoding round canbe performed during the PCR step of library preparation.

The oligonucleotides listed in Table 23 were used in the Examples above.“rG” is an RNA base and “₊G” is a locked nucleic acid base.

TABLE 23 Oligonucleotides Oligonucleotide Number Description SequenceSEQ ID NO. BC_0060 Round 3 barcode AGTCGTACGCCGATG SEQ ID NO. 16 linkerCGAAACATCGGCCAC BC_0062 PCR primer, used CAGACGTGTGCTCTTC SEQ ID NO. 17after template CGATCT switching (used with BC_0108) BC_0066Round 3 blocking GTGGCCGATGTTTCG SEQ ID NO. 18 strand CATCGGCGTACGACTBC_0076 Nextera Tagmentation CAAGCAGAAGACGGC SEQ ID NO. 19 PCR primerATACGAGATGATCTGG (TSBC07), sublibrary TGACTGGAGTTCAGACindex #1 (used with GTGTGCTCTTCCGATC BC_0118) T BC_0077Nextera Tagmentation CAAGCAGAAGACGGC SEQ ID NO. 20 PCR primerATACGAGATTCAAGTG (TSBC08), sublibrary TGACTGGAGTTCAGACindex #2 (used with GTGTGCTCTTCCGATC BC_0118) T BC_0078Nextera Tagmentation CAAGCAGAAGACGGC SEQ ID NO. 21 PCR primerATACGAGATCTGATCG (TSBC09), sublibrary TGACTGGAGTTCAGACindex #3 (used with GTGTGCTCTTCCGATC BC_0118) T BC_0079Nextera Tagmentation CAAGCAGAAGACGGC SEQ ID NO. 22 PCR primerATACGAGATAAGCTAG (TSBC10), sublibrary TGACTGGAGTTCAGACindex #4 (used with GTGTGCTCTTCCGATC BC_0118) T BC_0080Nextera Tagmentation CAAGCAGAAGACGGC SEQ ID NO. 23 PCR primerATACGAGATGTAGCC (TSBC11), sublibrary GTGACTGGAGTTCAG index #5 (used withACGTGTGCTCTTCCGA BC_0118) TCT BC_0081 Nextera TagmentationCAAGCAGAAGACGGC SEQ ID NO. 24 PCR primer ATACGAGATTACAAGG(TSBC12), sublibrary TGACTGGAGTTCAGAC index #6 (used withGTGTGCTCTTCCGATC BC_0118) T BC_0082 Nextera Tagmentation CAAGCAGAAGACGGCSEQ ID NO. 25 PCR primer ATACGAGATTTGACTG (TSBC13), sublibraryTGACTGGAGTTCAGAC index #7 (used with GTGTGCTCTTCCGATC BC_0118) T BC_0083Nextera Tagmentation CAAGCAGAAGACGGC SEQ ID NO. 26 PCR primerATACGAGATGGAACTG (TSBC14), sublibrary TGACTGGAGTTCAGACindex #8 (used with GTGTGCTCTTCCGATC BC_0118) T BC_0108 PCR primer, usedAAGCAGTGGTATCAAC SEQ ID NO. 27 after template GCAGAGTswitching (used with BC_0062) BC_0118 Nextera TagmentationAATGATACGGCGACC SEQ ID NO. 28 PCR primer N501 ACCGAGATCTACACTA(used with BC_0076 GATCGCTCGTCGGCA through BC_0083) GCGTCAGATGTGTATAAGAGACAG BC_0127 Template switching AAGCAGTGGTATCAAC SEQ ID NO. 29primer, HPLC purified GCAGAGTGAATrGrG+ (purchased from G Exigon) BC_0215Round 2 barcode CGAATGCTCTGGCCT SEQ ID NO. 30 linker CTCAAGCACGTGGATBC_0216 Round 2 blocking ATCCACGTGCTTGAGA SEQ ID NO. 31 strandGGCCAGAGCATTCG

Certain embodiments of this disclosure are described herein, includingthe best mode known to the inventors for carrying out the disclosure. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The applicants expect skilled artisans to employ suchvariations as appropriate, and the applicants intend for the variousembodiments of the disclosure to be practiced otherwise thanspecifically described herein. Accordingly, this disclosure includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the disclosure unless otherwise indicatedherein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above-citedreferences and printed publications are individually incorporated hereinby reference in their entirety.

It is to be understood that the embodiments of the present disclosureare illustrative of the principles of the present disclosure. Othermodifications that may be employed are within the scope of thedisclosure. Thus, by way of example, but not of limitation, alternativeconfigurations of the present disclosure may be utilized in accordancewith the teachings herein. Accordingly, the present disclosure is notlimited to that precisely as shown and described.

The particulars shown herein are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentdisclosure only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of various embodiments of thedisclosure.

It will be apparent to those having skill in the art that many changesmay be made to the details of the above-described embodiments withoutdeparting from the underlying principles of the disclosure. The scope ofthe present invention should, therefore, be determined only by thefollowing claims.

The invention claimed is:
 1. A method of uniquely labeling RNA moleculeswithin a plurality of cells, the method comprising: (a) fixing andpermeabilizing a first plurality of cells prior to step (b), wherein thefirst plurality of cells is fixed and permeabilized at below about 8°C.; (b) reverse transcribing the RNA molecules within the firstplurality of cells to form complementary DNA (cDNA) molecules within thefirst plurality of cells, wherein reverse transcribing the RNA moleculescomprises coupling reverse transcription (RT) primers to the RNAmolecules, wherein the RT primers comprise at least one of a poly(T)sequence or a random sequence; (c) dividing the first plurality of cellscomprising cDNA molecules into at least two primary aliquots, the atleast two primary aliquots comprising a first primary aliquot and asecond primary aliquot; (d) providing primary nucleic acid tags to theat least two primary aliquots, wherein the primary nucleic acid tagsprovided to the first primary aliquot are different from the primarynucleic acid tags provided to the second primary aliquot; (e) couplingthe cDNA molecules within each of the at least two primary aliquots withthe provided primary nucleic acid tags; (f) combining the at least twoprimary aliquots; (g) dividing the combined primary aliquots into atleast two secondary aliquots, the at least two secondary aliquotscomprising a first secondary aliquot and a second secondary aliquot; (h)providing secondary nucleic acid tags to the at least two secondaryaliquots, wherein the secondary nucleic acid tags provided to the firstsecondary aliquot are different from the secondary nucleic acid tagsprovided to the second secondary aliquot; (i) coupling the cDNAmolecules within each of the at least two secondary aliquots with theprovided secondary nucleic acid tags; (j) combining the at least twosecondary aliquots; (k) lysing the combined at least two secondaryaliquots of the first plurality of cells to release the cDNA moleculesfrom within the first plurality of cells to form a lysate; and (l)adding a protease inhibitor and a binding agent to the lysate such thatthe cDNA molecules bind the binding agent.
 2. The method of claim 1wherein before step (k), the method further comprises dividing thecombined at least two secondary aliquots into at least two finalaliquots, the at least two final aliquots comprising a first finalaliquot and a second final aliquot.
 3. The method of claim 1, whereinthe protease inhibitor comprises phenylmethanesulfonyl fluoride (PMSF)or 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). 4.The method of claim 1, further comprising: (m) conducting a templateswitch of the cDNA molecules bound to the binding agent; (n) amplifyingthe cDNA molecules to form an amplified cDNA molecule solution; and (o)introducing a solid phase reversible immobilization (SPRI) bead solutionto the amplified cDNA molecule solution, wherein the ratio of SPRI beadsolution to amplified cDNA molecule solution is about 0.7:1 or 0:8:1. 5.The method of claim 4, wherein a common adapter sequence is added to the3′-end of the released cDNA molecules by template switching.
 6. Themethod of claim 1, wherein the RT primers of step (b) further comprise afirst specific barcode, the method further comprising: (p) reversetranscribing RNA molecules within a second plurality of cells to formcDNA molecules within the second plurality of cells, wherein reversetranscribing the RNA molecules comprises coupling specific primers tothe RNA molecules from the second plurality of cells, wherein thespecific primers comprise a second specific barcode and at least one ofa poly(T) sequence or a random sequence, wherein the first specificbarcode is different from the second specific barcode such that the cDNAmolecules from the first plurality of cells can be identified incomparison to the cDNA molecules from the second plurality of cells; (q)dividing the second plurality of cells comprising cDNA molecules into atleast two primary aliquots, the at least two primary aliquots comprisinga first primary aliquot and a second primary aliquot; (r) providingprimary nucleic acid tags to the at least two primary aliquots, whereinthe primary nucleic acid tags provided to the first primary aliquot aredifferent from the primary nucleic acid tags provided to the secondprimary aliquot; (s) coupling the cDNA molecules within each of the atleast two primary aliquots with the provided primary nucleic acid tags;(t) combining the at least two primary aliquots; (u) dividing thecombined primary aliquots into at least two secondary aliquots, the atleast two secondary aliquots comprising a first secondary aliquot and asecond secondary aliquot; (v) providing secondary nucleic acid tags tothe at least two secondary aliquots, wherein the secondary nucleic acidtags provided to the first secondary aliquot are different from thesecondary nucleic acid tags provided to the second secondary aliquot;and (w) coupling the cDNA molecules within each of the at least twosecondary aliquots with the provided secondary nucleic acid tags.
 7. Themethod of claim 1, 4, or 5, wherein the RT primers further comprise a 5′overhang comprising a 5′ overhang sequence, and wherein each of thenucleic acid tags comprises: a first strand comprising: a barcodesequence comprising a 3′ end and a 5′ end; and a 3′ hybridizationsequence and a 5′ hybridization sequence flanking the 3′ end and the 5′end of the barcode sequence, respectively; and a second strandcomprising: a first portion complementary to the 5′ overhang sequence ofan RT primer or to the 5′ hybridization sequence of a previously couplednucleic acid tag; and a second portion complementary to the 3′hybridization sequence.
 8. The method of claim 7, further comprising:ligating at least two of the nucleic acid tags that are bound to thecDNA molecules.
 9. The method of claim 8, wherein the ligation isperformed within the first plurality of cells.
 10. The method of claim 1or 7, further comprising: ligating at least two of the nucleic acid tagsthat are bound to the released cDNA molecules.